CN120818618A

CN120818618A - System, method and application for detecting Staphylococcus aureus based on nucleic acid aptamers and B-PER-CRISPR signal amplification

Info

Publication number: CN120818618A
Application number: CN202511317741.7A
Authority: CN
Inventors: 张何; 彭媛; 马文杰; 蒋佳林; 傅昕; 罗家美; 杨宁
Original assignee: Hunan Institute of Engineering
Current assignee: Hunan Institute of Engineering
Priority date: 2025-09-16
Filing date: 2025-09-16
Publication date: 2025-10-21

Abstract

The present application provides a system, method, and application for detecting Staphylococcus aureus based on nucleic acid aptamers and B-PER-CRISPR signal amplification. Based on B-PER technology, the present application uses nucleic acid aptamers as recognition elements, Staphylococcus aureus as a target, and magnetic microspheres as probe carriers to combine the efficient trans-cleavage activity of CRISPR/Cas12a with B-PER technology to construct a signal amplification system. crRNA-Cas12a specifically binds to complementary sequences on linear DNA nanomolecules, and by cis-cleaving the bases of the complementary sequence on the linear DNA molecule, the crRNA-Cas12a-cleavage chain trimer is released from the linear DNA molecule. The trimer with trans-cleavage activity can non-specifically cleave a single-stranded DNA fluorescent probe. The fluorescent signal generated after the fluorescent probe breaks can be detected, achieving efficient detection of Staphylococcus aureus.

Description

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

Technical Field

The application relates to the technical field of label-free molecular probe colorimetric sensing, in particular to a system, a method and application for detecting staphylococcus aureus based on nucleic acid aptamer and B-PER-CRISPR signal amplification.

Background

Staphylococcus aureus (Staphylococcus aureus, abbreviated as staphylococcus aureus) is a pathogenic bacterium commonly found on human and animal skin and mucous membranes. As one of the main pathogens causing food-borne diseases and nosocomial infections, staphylococcus aureus has a very wide pathogenic range, can cause multiple system infections of epidermis, respiratory tract, blood flow and the like, and staphylococcus aureus toxin a is one of the important toxins causing food poisoning, and can cause severe poisoning symptoms and even death. The food is contaminated by staphylococcus aureus and can possibly produce enterotoxin when being stored for about 48 hours at the temperature of 20-37 ℃. Research shows that although 60 ℃ heating treatment for 1 hour can effectively kill staphylococcus aureus, enterotoxins produced by the staphylococcus aureus have thermal stability and still can keep activity, and influence on human bodies.

Currently, although plate counting, immunological and molecular biological methods are currently the dominant means of detecting staphylococcus aureus, their inherent drawbacks, such as long time-consuming incubation process, heavy detection effort, and limited sensitivity and specificity, limit the application efficacy of the above methods. Therefore, development of a rapid nucleic acid detection technology with high sensitivity and high specificity is an urgent need. However, the currently dominant PCR nucleic acid detection strategies have significantly short plates, are subject to laboratory contamination, are costly, are subject to false positives, are subject to sequence mismatches, and require the use of highly purified nucleic acid samples to ensure polymerase activity, which have limited their broad applicability.

Disclosure of Invention

In order to solve the technical problems, the scheme provides a system and a method for detecting staphylococcus aureus based on nucleic acid aptamer and B-PER-CRISPR signal amplification, and develops a novel technology for detecting staphylococcus aureus with simple operation, ultrasensitivity and high specificity. Based on the two-way primer exchange reaction technology, the application creatively combines CRISPR/Cas12a high-efficiency trans-cleavage activity with the two-way primer exchange reaction by taking the nucleic acid aptamer as an identification element, staphylococcus aureus as a target and magnetic microspheres as probe carriers, and constructs a novel signal amplification system.

Nucleic acid aptamers (aptamers) are single-stranded DNA or RNA molecules derived from ligand system evolution techniques that are capable of specifically recognizing and binding to a variety of corresponding target molecules. The nucleic acid aptamer is typically an oligonucleotide sequence centered in the 60.+ -.20 nt region in length. By folding to form a specific three-dimensional space structure, the target molecule is bound with high affinity, and the corresponding target is further identified. The aptamer is nucleic acid in nature, so the aptamer has the advantages of easiness in synthesis modification, good stability, no immunogenicity and the like. Depending on the type of target, the aptamers are DNA aptamers, which are more stable, and RNA aptamers, which are more structurally diverse. The aptamer has remarkable advantages in the field of microorganism detection, and compared with protein antibodies, the aptamer has the advantages of simpler preparation process, higher chemical modification flexibility and better physicochemical stability. These characteristics allow for efficient integration of the aptamer into the biosensing platform, thus achieving high specificity recognition and detection of microorganisms.

The bidirectional primer exchange reaction (B-PER) is an isothermal nucleic acid amplification technology participated by KF enzyme, target amplification is realized through autonomous circulation of two symmetrical primers (S1 and S2), the target triggers forward primer (S1) extension, a new chain T0 containing a reverse primer (S2) binding site is generated, after the T0 is combined with S2 and extended, T1 is released by replacement, and the T1 is combined with S2 to restart circulation immediately, so that a self-sustaining cascade reaction is formed. The technology can finish amplification in a short time under the condition of constant temperature.

CRISPR/Cas12A (original name Cpf1, now commonly referred to as Cas12A or FnCpf 1) is A representative system of the Type V-A subtype in the CRISPR-Cas family. Compared with other CRISPR systems (such as Cas9 and Cas 13), the unique characteristics of the CRISPR system enable the CRISPR system to exhibit unique advantages in the fields of gene editing, nucleic acid detection and the like. Cpf1 is made up of two parts, a Cas12a protein and a CRISPR array. CRISPR arrays comprise spacer sequences and repeat sequences, the latter derived from exogenous DNA fragments, for storing immune memory. The Cas12a protein acts as a multi-domain endonuclease capable of recognizing and cleaving the target DNA that can be base-complementarily paired with the spacer sequence. Unlike Cas9, cas12a only needs single-stranded guide RNA (sgRNA) guidance, generates 5' protruding sticky ends when DNA is cut, is more conducive to gene knock-in, and the smaller protein size is convenient for delivery, being widely used in the fields of gene editing, pathogen detection, biosensing, etc.

The mechanism of action of Cas12a is divided into adaptive (integration of exogenous DNA fragments), expression (crRNA maturation) and interference (targeting and trans-cleavage) phases. In the nucleic acid detection, the trans-cleavage activity is skillfully utilized, the target DNA triggers the Cas12a to cleave the reporter molecule (such as fluorescent labeled ssDNA), the visual signal amplification is realized, and the sensitivity can reach a single-molecule level. The CRISPR array transcribes to pre-crRNA, which is processed by nucleases to mature into sgRNA. The sgRNA can bind to Cas12a protein to form an RNP complex that possesses targeted cleavage activity. The targeted cleavage (cis-cleavage) RNP complex specifically cleaves double-stranded DNA (dsDNA) at a specific location upstream of PAM through the base complementary pairing sequence in the sgRNA and the target DNA. When the target DNA is cleaved, cas12a protein randomly cleaves surrounding ssDNA (single-stranded DNA), and this "side-cleavage" effect (trans-cleavage) can be used for signal amplification, which is a core principle of the nucleic acid detection technology using the Cpf1 system. The technical advantages include low off-target risk, multi-target editing capability and flexibility in adapting to different application scenarios.

In order to achieve the aim, the application firstly provides a system for detecting staphylococcus aureus based on nucleic acid aptamer and B-PER-CRISPR signal amplification, which comprises an arched bridge DNA probe, a ternary complex, crRNA, cas12a protein, dNTPs, a fluorescent probe and KF polymerase;

The arch bridge-shaped DNA probe is formed by base complementation pairing between a DNA single-chain S1 and a DNA single-chain S2, a ternary complex is formed by combining a nucleic acid aptamer, a connecting chain T and a magnetic microsphere in sequence, the nucleic acid aptamer can be specifically combined with staphylococcus aureus, a crRNA/Cas12a binary complex is formed by combining Cas12a protein and crRNA, dNTPs are selected from dATP, dCTP and dTTP;

The gene sequence of the nucleic acid aptamer is shown as SEQ ID NO.1, the gene sequence of the connecting chain T is shown as SEQ ID NO.2, the gene sequence of the DNA single strand S1 is shown as SEQ ID NO.3, the gene sequence of the DNA single strand S2 is shown as SEQ ID NO.4, the gene sequence of the crRNA is shown as SEQ ID NO.5, and the gene sequence of the fluorescent probe is TCCCCCCT.

Preferably, the partial sequences 5'-TTACCC-3', 5'-GGGTAA-3' in the DNA single strand S1 and the partial sequences 5'-TTACCC-3' in the DNA single strand S2 are in base complementary pairing, and the partial sequence 5'-GGGTAA-3' in the DNA single strand S1 and the partial sequence 5'-TTTTTT-3' in the DNA single strand S2 form a convex ring structure, so that the DNA single strand S1 and the DNA single strand S2 are combined to form a arched bridge DNA probe, namely a DAB arched bridge structure.

Based on a general inventive concept, the application also provides a method for detecting staphylococcus aureus for non-diagnosis and treatment purposes, which comprises the following steps of 1 to 6:

Preparing a arched bridge-shaped DNA probe, namely preparing freeze-dried powder of a DNA single strand S1 and a DNA single strand S2 into solutions by using DEPC water respectively, then mixing the two solutions, adding NEB buffer solution, reacting for 3-7 min at 90-100 ℃, and cooling to room temperature to obtain the arched bridge-shaped DNA probe.

Preferably, the step of preparing the freeze-dried powder of the DNA single strand S1 and the DNA single strand S2 into solutions by using DEPC water comprises preparing 100 mu M mother liquor by using DEPC water and diluting the mother liquor into 20 mu M diluent by using DEPC water. And then mixing and reacting with diluent to obtain the arch bridge-shaped DNA probe. Further, if the diluent is not used in time after being prepared, the diluent needs to be stored for standby at-25 ℃ to-18 ℃.

The room temperature in the application is 20-30 ℃.

And 2, preparing the nucleic acid aptamer diluent and the connecting chain T diluent, namely preparing the connecting chain T and the nucleic acid aptamer lyophilized powder into solutions by using HEPES buffer solution, and then adding BufferI buffer solution to obtain the connecting chain T diluent and the nucleic acid aptamer diluent.

And 3, preparing a ternary complex, namely mixing a diluent of a connecting chain T with the washed streptavidin magnetic microspheres, incubating the mixture on a magnetic separation frame at the oscillation speed of 280-320 r/min for 25-35 min at the temperature of 20-30 ℃, then magnetically separating and discarding supernatant, adding the diluent of the nucleic acid aptamer, continuously incubating the mixture at the oscillation speed of 280-320 r/min for 25-35 min at the temperature of 20-30 ℃, then adding DEPC water, carrying out vortex mixing, magnetically separating and discarding supernatant, and then adding DEPC water to resuspend the complex to obtain a ternary complex solution.

Preferably, the step of mixing the dilution of the linked chains T with the washed streptavidin magnetic microspheres is preceded by the steps of:

And re-suspending the streptavidin magnetic microspheres 25 s-35 s by using a vortex mixer, then sucking part of the re-suspended solution containing the streptavidin magnetic microspheres into an EP tube, placing the EP tube on a magnetic separation frame, standing, discarding the supernatant after the streptavidin magnetic microspheres are adsorbed, adding BufferI buffer solution, fully mixing again by using the vortex mixer, separating again by using the magnetic separation frame and discarding the supernatant, adding BufferI buffer solution again, fully mixing by using the vortex mixer, separating by using the magnetic separation frame and discarding the supernatant, and obtaining the washed streptavidin magnetic microspheres.

Preferably, in the step 3, the molar ratio of the connecting chain T to the nucleic acid aptamer to the streptavidin magnetic microsphere is 1 (1.8-2.0) (0.9-1.2).

Preferably, the ternary complex solution may be stored at 2-5 ℃ prior to use.

And 4, performing a two-way primer exchange reaction, namely mixing a sample to be detected with a ternary complex solution and DEPC water, removing supernatant after magnetic separation, adding an arch bridge-shaped DNA probe, KF enzyme, dCTP, dTTP, dATP and a Buffer solution with 10 times of concentration of the KF enzyme for mixing, performing a reaction at 35-40 ℃ for 60-120 min, performing a reaction at 70-80 ℃ for 25-35 min, inactivating the KF enzyme, and washing for 3-4 times by using a PBS Buffer solution to obtain a solution after the two-way primer exchange reaction.

Preferably, in step 4, the ternary complex is present in the reaction solution at a concentration of 480nM to 520nM. More preferably 500nM.

Preferably, the molar ratio of the ternary complex to the arched bridge DNA probe is 1 (0.8-1.2).

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

Preferably, the concentration of KF enzyme in the reaction solution is 0.02U/. Mu.L to 0.04U/. Mu.L.

Preferably, the concentration of staphylococcus aureus in the sample to be tested is more than or equal to 13 CFU/mL.

Preferably, the reaction time is 85 min-95 min under the condition of 35-40 ℃.

It is further preferred that the reaction is carried out for 90min at 37 ℃ and then for 30min at 75 ℃.

And 5, trans-cutting a single-chain fluorescent probe by a cis-cutting product chain of the Cas12a protein, namely adding DEPC water, the Cas12a protein, crRNA, the fluorescent probe and a Buffer solution with 10 times concentration of the Cas12a into the solution after the two-way primer exchange reaction, reacting for 55-65 min at 35-40 ℃, heating to 70-80 ℃, and preserving heat for 4-6 min to inactivate the Cas12a protein.

Preferably, DEPC water, cas12a protein, crRNA, fluorescent probe, and Buffer at 10-fold concentration of Cas12a are added to the solution after the two-way primer exchange reaction, reacted at 37 ℃ for 60min, and then warmed to 75 ℃ for 5min.

And 6, detecting and analyzing by using a fluorescence spectrophotometer, namely adding DEPC water, detecting by using the fluorescence spectrophotometer, and collecting fluorescence emission spectra under a specific excitation wavelength.

Based on a general inventive concept, the application also provides an application of the system for detecting staphylococcus aureus in food based on nucleic acid aptamer and B-PER-CRISPR signal amplification.

Preferably, the foodstuff comprises milk.

The detection mechanism of the application is as follows:

As shown in FIG. 1, the arched bridge DNA probe (DAB) is formed by mixing two single-stranded DNAs of S1 and S2, under specific conditions, the S1 sequence (a 'part sequence 5' -TTACCC-3 'and a part sequence 5' -GGGTAA-3 ') and the S2 sequence (a' part sequence 5'-TTACCC-3' and a part sequence 5 '-GGGTAA-3') are in base complementary pairing, a convex ring structure (c part sequence 5 '-TTTTTT-3') is formed in the middle, so that the arched bridge DNA probe (DAB) is obtained, and Primer Exchange Reaction (PER) can be carried out on both ends (b part sequence 5 '-CACCGTTA-3') and a part sequence 5 '-GGGTAA-3') of the obtained product DAB.

The streptavidin is high-affinity biotin and can capture the connecting chain T, and the base complementary pairing of the connecting chain T is combined with the nucleic acid aptamer, so that an acid aptamer/connecting chain T/streptavidin magnetic microsphere ternary complex is formed. Meanwhile, the ternary complex can seal the function of a target recognition region of the aptamer, and when no target exists in a system, the aptamer-connecting chain T double-chain structure is stable, and the subsequent bidirectional primer exchange reaction (B-PER) is blocked. When staphylococcus aureus exists, the aptamer preferentially performs competitive binding with a bacterial surface target, dissociates from the connecting chain T, releases the free connecting chain T, and can start the B-PER cascade reaction. The base (5 '-TAACGGTG-3') of the T part of the connecting strand can complementarily bind to the b sequence part (5 '-CACCGTTA-3') of the right end of DAB and can also be used as a primer of right PER. When KF polymerase and dNTPs are present, the ligation strand T is extended and stopped at the designed termination sites (5 '-GGGCC-3', 5 '-CCCC-3', 5'-GGGGG-3', 5 '-GGCCC-3'), and sequence amplification is achieved, with the original ligation strand T being extended to a new trigger strand T0.

The termination sites set in the present application are controlled by three nucleotides dATP, dCTP, dTTP. Specifically, the DNA chain extension is terminated because the raw material lacks guanine (G), the cytosine (C) on the corresponding template strand (S1 or S2) cannot be amplified correspondingly, the DNA strand cannot continue to extend, and the 3' -end of DAB is modified with an inverted dT to prevent its amplification or cleavage of the DNA strand. In addition, the released trigger strand T0 can be paired with a partial region at the left end of DAB and used as a primer to participate in a primer exchange reaction, so that the growth of a corresponding repeated sequence is promoted, and DAB is recycled. Finally, a DNA single strand (product strand) containing a large number of repeated sequences is obtained.

The product chain formed by the B-PER contains a large number of repeated sequences, wherein every three repeated sequences (total 18 bases) can be complementarily paired and combined with the designed crRNA, and the magnetic microsphere is arranged on the product chain, so that the magnetic separation and purification are convenient. After adding Cas12a protein and crRNA to the product strand, the crRNA recognizes three repeated sequences of the product strand (the three repeated sequences are base complementary paired with one crRNA) to trigger Cas12a-crRNA complex, activate its cis-cleavage activity, and specifically cleave the activation strand (ssDNA). The cleavage product can form crRNA/Cas12 a/cleavage chain trimer, and the active complex opens the trans-cleavage function by allosteric effects, non-specifically degrading the fluorescent reporter. Trans-cleavage causes the fluorescent group (F) and the quenching group (Q) to be distant from each other and the fluorescent signal to be recovered.

Compared with the prior art, the application has the following beneficial effects:

The system is used for constructing a one-tube type constant temperature system based on aptamer specific recognition, B-PER amplification and CRISPR/Cas12a signal amplification to detect staphylococcus aureus in food, so that high-sensitivity and high-specificity detection performance is realized. Specifically:

(1) The high sensitivity is realized by constructing an amplification system based on CRISPR/Cas12a high-efficiency trans-cleavage activity and bidirectional primer exchange reaction amplification, wherein a product chain containing a large number of repeated sequences generated by the bidirectional primer exchange reaction can be combined with a complementary sequence of crRNA/Cas12a dimer (the combination mode is that three repeated sequences are complementary and paired with a sequence of crRNA), the nuclease domain of the combined Cas12a is activated, single-stranded DNA combined with crRNA/Cas12a protein is cis-cleaved, crRNA/Cas12 a/cleavage chain trimer is released after cleavage, trans-cleavage activity is activated, a DNA fluorescent probe is cut off by trans-cleavage, a fluorescent group F is far away from a quenching group Q, and a fluorescent signal is recovered.

(2) The realization of high specificity is that the nucleic acid aptamer has the capability of specifically recognizing a target, the nucleic acid aptamer (5'-CACCGCCACCGTGCTACAAC-3') can be complementarily matched and combined with a connecting chain T (5'-GTTGTAGCACGGTAACGGTG-3') on the surface of the magnetic microsphere, and the active area of the nucleic acid aptamer is blocked to prevent a higher background from being generated under the condition of no target. The S1 part segment (5 '-CACCGTTA-3') of the DNA arch bridge structure can be combined with the connecting chain T part segment (5 '-TAACGGTG-3'), after the nucleic acid aptamer is separated from the connecting chain T, the DNA arch bridge structure is combined with the connecting chain T to carry out B-PER reaction, so that the connecting chain T is amplified into a product chain, the product chain on the surface of the magnetic microsphere can be obtained through magnetic separation, and the subsequent CRISPR reaction is carried out.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the embodiments of the present application will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present application, and other drawings may be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 is a schematic diagram of a Staphylococcus aureus detection system according to the present application.

FIG. 2 is a graph showing the results of the feasibility of the method for detecting Staphylococcus aureus of example 2.

FIG. 3 is a graph showing the specificity of the Staphylococcus aureus detection system of example 3 for a strain.

FIG. 4 is a graph showing the results of the test under different KF enzyme concentration conditions in example 4.

FIG. 5 is a graph showing the results of the detection under different B-PER reaction time conditions in example 5.

FIG. 6 is a graph showing the results of the test under the conditions of different concentrations of the aptamer-linker T-modified magnetic microsphere in example 6.

FIG. 7 is a graph of fluorescence spectroscopy analysis of example 7 for detection of different concentrations of Staphylococcus aureus.

FIG. 8 is a graph of signal saturation for detection of Staphylococcus aureus at various concentrations in example 7.

FIG. 9 is a calibration curve for Staphylococcus aureus at various concentrations for example 7.

Detailed Description

The examples described in this specification are for the purpose of illustrating the application only and are not intended to limit the application.

For simplicity, only a few numerical ranges are explicitly disclosed. However, any lower limit may be combined with any upper limit to form a range not explicitly recited, and any lower limit may be combined with any other lower limit to form a range not explicitly recited, as may any upper limit combined with any other upper limit. Furthermore, each point or individual value between the endpoints of the range is included within the range, although not explicitly recited. Thus, each point or individual value may be combined as a lower or upper limit on itself with any other point or individual value or with other lower or upper limit to form a range that is not explicitly recited.

The term "%" as used herein refers to the mass percent unless otherwise specified, but the term "percent" as used herein refers to the gram of solute contained in a solution of 100 mL unless otherwise specified.

The parts by weight described in the present application may be those known in the art such as mu g, mg, g, kg, or may be multiples thereof such as 1/10, 1/100, 10 times, 100 times, etc.

The application is further illustrated below with reference to examples. It should be understood that these embodiments are for illustrative purposes only, as various modifications and changes in light thereof will be suggested to persons skilled in the art and are to be included within the scope of this disclosure. Unless otherwise indicated, all parts, percentages and ratios reported in the examples below are on a mass basis, and all reagents used in the examples are commercially available or were obtained synthetically according to conventional methods and can be used directly without further treatment, as well as the instruments used in the examples.

The oligonucleotide chains and related chemical reagents according to the present application were purchased from Shanghai Biotechnology Co., ltd, as shown in Table 1.

Wherein, the nucleotide sequence underlined and inclined in the sequence of the connecting chain T is combined with the DAB arch bridge structural part sequence, the sequence of the thickened part in S1 and S2 represents a convex ring domain for forming the DAB arch bridge, the part of the nucleotide sequence underlined and inclined in S1 is combined with the connecting chain T after the DAB arch bridge is formed, and the part of the sequence underlined in crRNA is a product chain combining part formed by the B-PER.

Example 1 detection of Staphylococcus aureus based on a Signal amplification System of nucleic acid aptamer and B-PER-CRISPR

(1) Pretreatment of DNA sequences, constructs

S1, S2 solution preparation, namely preparing the freeze-dried powder of the S1 and S2 sequences into 100 mu M concentrated solution by using diethyl pyrocarbonate (DEPC), respectively taking 10 mu L of each solution into a PCR tube, adding 40 mu L of DEPC water to dilute the solution into 20 mu M solution, and preserving at-20 ℃.

Linker T, nucleic acid aptamer probes linker T, nucleic acid aptamer lyophilized powder was prepared as 100. Mu.M concentrated solution with 4- (2-hydroxyethyl) piperazine-1-ethane sulfonic acid (HEPES) buffer (20mM HEPES,20mM NaCl,8mM MgCl ₂•6H₂ O,1mM KCl, pH=7.5). 10 mu L of the concentrated solution of the connecting chain T is taken in a PCR tube, 90 mu L BufferI buffer solution (1M NaCl, 1mM Na ₂ EDTA, 10mM Tris-HCl, 0.01% -0.1% Tween-20 and pH=7.5) is added to dilute the solution into 10 mu M solution, 10 mu L of the concentrated solution of the aptamer is taken in the PCR tube, and 40 mu L BufferI buffer solution is added to dilute the solution into 20 mu M solution. All were kept at-20℃until use.

The crRNA and fluorescent probe solution is prepared by preparing 100 mu M concentrated solution from DEPC water, 10 mu L of the crRNA concentrated solution is taken and placed in a PCR tube, 90 mu L of DEPC water is added to dilute the crRNA and fluorescent probe solution into 10 mu M solution, the temperature is minus 20 ℃, the preservation is carried out, 10 mu L of the fluorescent probe concentrated solution is taken and placed in 90 mu L of DEPC water in an opaque centrifuge tube, and 10 mu M fluorescent probe solution is prepared, and the temperature is minus 20 ℃ and the preservation is carried out in a dark place.

(2) Staphylococcus aureus pretreatment

Resuscitating strain was inoculated into beef extract peptone liquid medium (5 g NaCl, 3g beef extract, 10g peptone), and cultured under shaking at 37℃and 220 rpm. 1mL of the bacterial liquid was centrifuged at 300r/min for 5min, the supernatant was discarded, and the bacterial pellet was washed twice with PBS 1 Xbuffer (137 mM NaCl ₂、7mM KCl、10mM Na₂HPO₄、1.8mM KH₂PO₄, pH=7.4), resuspended in 1mL of PBS 1 Xsolution, and stored at 4℃for further use. Viable bacteria count adopts a 10-fold gradient dilution method, 100 mu L of dilution liquid is coated on a beef extract peptone plate, colony count is counted after incubation for 24 hours at 37 ℃, and the viable bacteria concentration is expressed by CFU/mL.

(3) Formation of DNA Arch Structure

Mu. L S1 (20. Mu.M) and 5. Mu. L S2 (20. Mu.M) solutions and 10. Mu.L NEB buffer 2 (10X, 10mM MgCl _、 mM DTT, 50mM NaCl, 10mM Tris-HCl, pH=7.9) were added to 80. Mu.L DEPC water. The mixture was heated to 95 ℃ in a water bath and maintained at this temperature for 5min, then cooled to room temperature to give DAB which was stored at-20 ℃ for later use in experiments.

(4) Nucleic acid aptamer-connecting chain T modified magnetic microsphere solution

After 30 seconds of re-suspending the streptavidin magnetic microspheres with a vortex mixer, 50 μl (10 μΜ) was pipetted into the EP tube. Then, it is placed on a magnetic separation frame, left for a while, after the magnetic microspheres are adsorbed, the supernatant is discarded. 100 mu L BufferI was added and thoroughly mixed again using a vortex, separated again by means of a magnetic separation rack and the supernatant discarded, and the washing step was repeated once. Next, 50. Mu.L (10. Mu.M) of the ligation chain T (diluted with BufferI solution) was added, incubated at 25℃for 30 minutes at a shaking speed of 300r/min, followed by magnetic separation and discarding the supernatant. Thereafter, 100. Mu.L of DEPC water was added, vortexed, magnetically separated and the supernatant discarded, and this washing step was repeated once. Then 50. Mu.L (20. Mu.M) of aptamer (diluted with BufferI solution) was added and incubation was continued with shaking at 25℃for 30 minutes at 300 r/min. Thereafter, 100. Mu.L of DEPC water was added repeatedly, vortexed and mixed well, and the steps of magnetically separating and discarding the supernatant were performed once, and the final washing was performed. Finally, 50 μl of DEPC water was added to resuspend the magnetic microspheres to obtain a solution of aptamer-linker-T modified magnetic microspheres, which was stored at 4 ℃ for later use in experiments.

(5) B-PER reaction

Mu.L of a sample to be tested and 5. Mu.L of a solution of nucleic acid aptamer-linker T modified magnetic microsphere (1. Mu.M) were added to an EP tube, 7.5. Mu.L of DEPC water was added, 20. Mu.L of supernatant was removed after magnetic separation, 5. Mu.L of DAB (1. Mu.M), 2. Mu.L of KF enzyme (0.5U/. Mu.L), 2.5. Mu.L of KF enzyme Buffer (10X), 3. Mu.L of dNTPs (each of 10mM dCTP, dTTP, dATP) were added to the tube, and after reaction at 37℃for 90min and 75℃for 30min in a water bath, the KF enzyme was inactivated, washed three times with PBS 1 Xbuffer, and finally 10. Mu.L of reaction solution was obtained.

(6) Cas12a protein cis-cleavage product strand trans-cleavage single-stranded fluorescent probe and resultant fluorescence analysis

Mu.L of DEPC water, 3. Mu.L of Cas12a (1. Mu.M), 3. Mu.L of Cas12a Buffer (10X), 3. Mu.L of crRNA (10. Mu.M) and 2. Mu.L of fluorescent probe (10. Mu.M) were taken and added to an EP tube containing the above reaction solution (10. Mu.L), reacted at 37℃for 60min in the absence of light, followed by inactivation of Cas protein at 75℃in a water bath for 5 min. 170 mu L of DEPC water is added into an EP tube, the reaction system is uniformly mixed by vortex oscillation, a fluorescence emission spectrum is acquired by using a fluorescence spectrophotometer under the excitation wavelength of 480nm, the maximum absorption peak at F520nm can be obtained, and the numerical value is recorded.

EXAMPLE 2 feasibility of the detection method for Staphylococcus aureus

To verify the feasibility of the aptamer and B-PER-CRISPR system to detect staphylococcus aureus, 5 sets of key control experiments (controlled by a single variable) were set up, the detection feasibility analysis results of which are shown in figure 2. In FIG. 2, the curve from top to bottom is that curve 1 is a normal experiment group, namely, the method of example 1 is adopted to directly replace a sample to be tested with staphylococcus aureus bacterial liquid, curve 2 is that Cas12a protein is absent, other conditions and factors are unchanged except DEPC water with corresponding volume is added, curve 3 is that KF enzyme is not added, DEPC water with corresponding volume is added, other conditions and factors are not changed, curve 4 is that crRNA is not added, DEPC water with corresponding volume is added, other conditions and factors are unchanged, and curve 5 is a blank group, namely, staphylococcus aureus bacterial liquid is not added, DEPC water with corresponding volume is replaced, and the experiment system is complete.

Curve 1 can normally recognize a target, trigger an aptamer to release a connecting strand T, enter B-PER, and the generated product strand is in base complementary pairing with crRNA, so that a fluorescent probe can be cut in a trans-form, and a fluorescent signal is recovered. Curve 2 has no Cas12a protein, cannot form an active cleavage complex, has lost trans-cleavage function, and has no fluorescent signal. Curve 3 does not have KF enzyme, can not carry out B-PER reaction in a large amount, has insufficient product chain generation and can not generate a large amount of fluorescent signals. Curve 4 fails to target Cas12a due to crRNA deletion, resulting in failure of trimer assembly, and no fluorescent signal. Curve 5 does not add a target, the connecting strand T is blocked by a nucleic acid aptamer, and therefore the DNA arch bridge structure cannot bind to the connecting strand T, resulting in incapacity of cleaving the fluorescent probe and low fluorescence intensity.

Example 3 specificity analysis of Staphylococcus aureus detection System for target

To evaluate the specificity of the detection system to staphylococcus aureus, a fluorescent signal was detected at F520nm under optimal conditions using vibrio parahaemolyticus, salmonella, three microbial mixtures and a blank as controls. The results are shown in FIG. 3, which shows that Salmonella and E.coli fluorescence values are near the blank level, while Staphylococcus aureus and mixtures thereof exhibit significantly enhanced signals. The result shows that the detection system based on the aptamer has good specificity and recognition capability on target bacteria (staphylococcus aureus).

Example 4 KF search for optimal concentration of enzyme

To ensure that the single stranded DNA (ssDNA) product generated by the B-PER reaction is of sufficient length to meet the requirements of the downstream CRISPR/Cas12a system for efficient recognition and cleavage of the target sequence. This example systematically optimizes the concentration of the key elongase Klenow Fragment (exo-) (KF enzyme). Considering that the concentration of KF enzyme directly affects the efficiency of primer extension and the length distribution of product strand in isothermal amplification, the present example sets up KF enzyme concentration gradient experiment groups (0.01U/. Mu.L, 0.02U/. Mu.L, 0.04U/. Mu.L, 0.06U/. Mu.L) covering different activity units, and each experiment was observed to detect fluorescent signal at F520nm, and the results are shown in FIG. 4. As can be seen from FIG. 4, the fluorescence signal reaches a maximum value at a KF enzyme concentration of 0.04U/. Mu.L. Thus, the KF enzyme concentration under the optimal reaction conditions was 0.04U/. Mu.L.

Example 5B-PER reaction optimum reaction time exploration

The reaction time is a key kinetic parameter affecting the efficiency of the B-PER reaction. To ensure sufficient binding of nucleic acids and to achieve optimal detection performance, the present example systematically conducted a condition optimization experiment of the reaction time parameters. Fluorescence signals at F520nm were observed for each experiment by setting up a gradient panel covering different B-PER reaction durations (30 min, 60 min, 90min, 120 min, 150 min). As a result, as shown in FIG. 5, the fluorescence signal was maximized when the reaction time was 90min. Thus, the optimal reaction time was determined to be 90min.

Example 6 optimal concentration exploration of aptamer-linker T-modified magnetic microsphere solutions

To optimize the efficiency of use and reduce unnecessary loss of the aptamer-linker T-modified magnetic microspheres (i.e., probes), this example systematically performed a conditional optimization of the key parameter, probe concentration. By setting a plurality of groups of experimental conditions covering different probe concentration gradients (250 nM, 500nM, 1000nM, 2000 nM), the law of influence of probe concentration on the performance of the probe in a target body is accurately explored. The results are shown in FIG. 6. The fluorescence signal reached a maximum at a probe concentration of 500nM. The probe concentration under optimal reaction conditions was 500nM.

Example 7 detection and analysis of different concentrations of bacterial species

Under the optimal system of optimal KF enzyme concentration, B-PER reaction time length and nucleic acid aptamer-connecting chain T modified magnetic microsphere, an experimental group for detecting staphylococcus aureus suspension (10 ²~10¹⁰ CFU/mL) with different concentrations based on a signal amplification system of the nucleic acid aptamer and the B-PER-CRISPR is arranged, and the detection fluorescence signals of the experimental group are observed. Fig. 7 is a graph of fluorescence spectrum analysis for detection of staphylococcus aureus suspensions of different concentrations, where the fluorescence spectrum intensity increases with increasing bacterial concentration for a corresponding concentration ：10¹⁰CFU/mL、10⁹CFU/mL、10⁸CFU/mL、10⁷CFU/mL、10⁶CFU/mL、10⁵CFU/mL、10⁴CFU/mL、10³CFU/mL、10²CFU/mL; of staphylococcus aureus suspension from top to bottom. FIG. 8 is a graph of saturation of detection signals from staphylococcus aureus at 520nm at different concentrations. As can be seen from fig. 8, when the staphylococcus aureus concentration was below 1.0×10 ⁶ CFU/mL, the fluorescence signal at 520nm was positively correlated with the bacterial concentration (R ² =0.998). Fig. 9 is a calibration curve for staphylococcus aureus at different concentrations. As can be seen from fig. 9, the staphylococcus aureus concentration exhibited a significant linear correlation between 10 ²~10⁶ CFU/mL, and the curve regression equation was Δf _{520 nm} =571.3logc+3210.4 (r2= 0.9976), where Δf _520nm represents the relative fluorescence intensity at 520nm and log c (CFU/mL) was the logarithm of the staphylococcus aureus concentration. The relative fluorescence intensity values were linearly related to the logarithm of the staphylococcus aureus concentration in the range of 10 ² to 10 ⁶ CFU/mL, with a detection limit of 13CFU/mL (calculation standard: lod=3σ/S; σ is standard deviation of blank solution, S is linear slope).

Example 8 detection of Staphylococcus aureus in milk

In order to examine the detection performance of the method in the detection of the actual sample, 10-fold diluted milk is used as a matrix, and staphylococcus aureus is added to evaluate the detection performance of the aptamer and the B-PER-CRISPR system in the actual sample.

The actual sample is prepared by adding a staphylococcus aureus bacterial liquid containing 1.0X10 ²、1.0×10³ and 1.0X10 ⁴ CFU/mL as a mother liquid into sterilized pure milk.

The experimental results are shown in Table 2, and the average recovery rate of the actual sample detection is 98.9% -100.7%. The method has better detection performance on staphylococcus aureus in milk samples.

While the application has been described with reference to certain preferred embodiments, it will be understood by those skilled in the art that various changes and substitutions of equivalents may be made and equivalents will be apparent to those skilled in the art without departing from the scope of the application. Therefore, the protection scope of the application is subject to the protection scope of the claims.

Claims

1. A system for detecting staphylococcus aureus based on nucleic acid aptamer and B-PER-CRISPR signal amplification is characterized by comprising an arched bridge DNA probe, a ternary complex, crRNA, cas12a protein, dNTPs, a fluorescent probe and KF polymerase;

The arch bridge-shaped DNA probe is formed by base complementation pairing between a DNA single-chain S1 and a DNA single-chain S2 through partial sequences, the ternary complex is formed by sequentially combining a nucleic acid aptamer, a connecting chain T and magnetic microspheres, the nucleic acid aptamer can be specifically combined with staphylococcus aureus, and dNTPs are selected from dATP, dCTP and dTTP;

2. A method for detecting staphylococcus aureus for non-diagnostic purposes, comprising the steps of:

Preparing a arched bridge-shaped DNA probe, namely preparing freeze-dried powder of a DNA single strand S1 and a DNA single strand S2 into solutions by using DEPC water respectively, then mixing the two solutions, adding NEB buffer solution, reacting for 3-7 min at 90-100 ℃, and cooling to room temperature to obtain the arched bridge-shaped DNA probe;

preparing a nucleic acid aptamer diluent and a connecting chain T diluent, namely preparing a solution from a connecting chain T and nucleic acid aptamer lyophilized powder by using a HEPES buffer solution, and then adding BufferI buffer solutions to obtain the diluent of the connecting chain T and the diluent of the nucleic acid aptamer;

Step 3, ternary complex preparation, namely mixing the diluent of the connecting chain T with washed streptavidin magnetic microspheres, incubating the mixture on a magnetic separation frame at the oscillating speed of 280-320 r/min for 25-35 min at the temperature of 20-30 ℃, then magnetically separating and discarding supernatant, adding the diluent of the nucleic acid aptamer, continuously incubating the mixture at the oscillating speed of 280-320 r/min for 25-35 min at the temperature of 20-30 ℃, then adding DEPC water, uniformly vortex, magnetically separating and discarding supernatant, and then adding the DEPC water to resuspend the complex to obtain ternary complex solution;

Step 4, a two-way primer exchange reaction, namely mixing a sample to be detected with the ternary complex solution and DEPC water, removing supernatant after magnetic separation, adding the arch bridge-shaped DNA probe, KF enzyme, dCTP, dTTP, dATP and Buffer with 10 times of concentration of KF enzyme for mixing, reacting for 60-120 min at 35-40 ℃, reacting for 25-35 min at 70-80 ℃ and inactivating the KF enzyme, washing for 3-4 times by using PBS Buffer to obtain a solution after the two-way primer exchange reaction;

Step 5, a single-chain fluorescent probe is cut in a trans way by a cis-cleavage product chain of Cas12a protein, namely DEPC water, cas12a protein, crRNA, fluorescent probe and Buffer solution with 10 times concentration of Cas12a are added into the solution after the two-way primer exchange reaction, the reaction is carried out for 55 min-65 min under the condition of 35 ℃ to 40 ℃, then the temperature is raised to 70 ℃ to 80 ℃ and kept for 4 min-6 min, and Cas12a protein is inactivated;

Step 6, fluorescence spectrophotometer detection analysis, namely adding DEPC water, uniformly mixing, detecting by using the fluorescence spectrophotometer, and collecting fluorescence emission spectrum under specific excitation wavelength;

3. The method according to claim 2, wherein in the step 3, before mixing the diluted solution of the link chain T with the washed streptavidin magnetic microspheres, comprising the steps of:

Suspending the streptavidin magnetic microspheres 25 s-35 s by a vortex mixer, then sucking part of the solution containing the streptavidin magnetic microspheres after the suspension into an EP tube, placing the EP tube on a magnetic separation frame, standing, discarding the supernatant after the streptavidin magnetic microspheres are adsorbed, then adding BufferI buffer solution, fully mixing again by the vortex mixer, separating again by the magnetic separation frame and discarding the supernatant, adding BufferI buffer solution again, fully mixing by the vortex mixer, separating by the magnetic separation frame and discarding the supernatant, and obtaining the washed streptavidin magnetic microspheres.

4. A method according to any one of claims 2 to 3, wherein in step 4, the molar ratio of the ternary complex to the arched DNA probe is 1 (0.8 to 1.2).

5. The method according to any one of claims 2 to 3, wherein in step 4, the concentration of KF enzyme in the reaction solution is 0.02U/. Mu.L to 0.04U/. Mu.L.

6. A method according to any one of claims 2 to 3, wherein in step 4, the concentration of staphylococcus aureus in the sample to be tested is not less than 13 CFU/mL.

7. A method according to any one of claims 2 to 3, wherein in step 4, the reaction time at 35 ℃ to 40 ℃ is 85min to 95min.

8. A method according to any one of claims 2 to 3, wherein in step 4, the ternary complex has a concentration of 480nm to 520nm in the reaction solution.

9. A method according to any one of claims 2 to 3, wherein the excitation wavelength is 480nm.

10. The use of a system for detecting staphylococcus aureus according to claim 1 or a method for detecting staphylococcus aureus according to any one of claims 2 to 9 for non-diagnostic purposes in a foodstuff, wherein the foodstuff comprises milk.