Method for detecting staphylococcus aureus

Abstract

The application provides an adsorption magnetic bead. The adsorption magnetic bead comprises: a Ni-polymethacrylate magnetic bead; and a binding protein used for binding Staphylococcus aureus, wherein the binding protein is connected to the Ni-polymethacrylate magnetic bead. In the application, the binding protein in the adsorption magnetic bead can bind Staphylococcus aureus, the adsorption magnetic bead is added into a sample to be detected, Staphylococcus aureus is bound by the binding protein, and then the adsorption magnetic bead can capture and enrich Staphylococcus aureus, which is helpful for subsequent detection of Staphylococcus aureus. The adsorption magnetic bead, the kit and the detection method of Staphylococcus aureus provided by the application have the advantages of reduced strain culture steps, short detection time and improved detection efficiency.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology. Specifically, this invention relates to a method for detecting Staphylococcus aureus. More specifically, this invention relates to an adsorption magnetic bead and its uses, a reagent kit and its uses, and a method for detecting Staphylococcus aureus. Background Technology

[0002] Staphylococcus aureus (S. aureus) is widely distributed in nature and easily contaminates foods such as milk and vegetables, making it one of the leading pathogens causing infections in humans and animals. Staphylococcus aureus produces virulence factors, including enterotoxins, exfoliative toxins, toxic shock syndrome toxin-1, and ubiquitin-valentin leukocytes associated with toxic shock syndrome, food poisoning, severe allergic diseases, and autoimmune diseases. Reports indicate that Staphylococcus aureus is widely present in raw milk and dairy products. In recent years, Staphylococcus aureus has become the third leading foodborne microbial pathogen after Salmonella and Vibrio parahaemolyticus. Due to the lack of effective, rapid, and visual detection methods, food poisoning incidents caused by Staphylococcus aureus occur frequently.

[0003] Therefore, there is an urgent need to establish a rapid, sensitive detection method that can be applied in large quantities. Summary of the Invention

[0004] The present invention aims to at least partially address one of the technical problems existing in the prior art. To this end, the present invention provides a method for detecting Staphylococcus aureus, which can effectively detect Staphylococcus aureus.

[0005] This invention is based on the following discoveries of the inventors:

[0006] Several rapid methods for detecting and identifying Staphylococcus aureus in food samples have been developed, such as polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), and nucleic acid-based molecular biology methods. These advanced microbial detection methods have reduced detection time from days to hours, but they still have some limitations. For example, these methods are often expensive, require high-tech laboratory equipment and skilled technicians, and cannot be used for on-site testing. Furthermore, in remote areas, the lack of laboratory equipment severely limits the detection of foodborne pathogens.

[0007] Bacteriophages are viruses that specifically infect bacteria; also known as "bacterial viruses," they typically range in size from 20 to 200 nm, have a short lifespan (approximately 20 to 60 minutes), and are widely recognized as the most abundant biological entity on Earth, numbering approximately 10^10^6. 31Within bacteria, there may also be corresponding bacteriophages, which play a crucial role in the ecological balance of microbial life. The main structure of a bacteriophage can be divided into a nucleocapsid or head, a complex tail structure, and a base. The head, capsid, and tail are composed of proteins, with the head capsid containing the bacteriophage's genetic material, nucleic acid (DNA or RNA). The capsid attaches to a fibrous tail, used to recognize and adhere to receptors on the bacterial cell surface.

[0008] Immunomagnetic separation (IMS) is a technique that combines immunology with magnetic beads. The principle is to prepare immunomagnetic beads by binding specific antibodies to magnetic beads, which are then combined with specific antigens in a mixed solution. Through magnetic separation, the target antigen is completely separated from other impurities, achieving enrichment and separation. Compared with traditional methods, it is not only faster and simpler, but also improves the sensitivity and accuracy of detection.

[0009] Based on this, in one aspect of the present invention, an adsorption magnetic bead is provided. According to an embodiment of the present invention, the adsorption magnetic bead comprises: Ni-polymethyl methacrylate magnetic beads; and a binding protein for binding Staphylococcus aureus, wherein the Ni-polymethyl methacrylate magnetic beads and the binding protein are linked.

[0010] The inventors also discovered that the binding protein in the magnetic beads can bind (especially specifically bind) Staphylococcus aureus. When these magnetic beads are added to the sample to be tested, the binding protein binds to Staphylococcus aureus, and further, magnetic separation is used to capture and enrich the Staphylococcus aureus, which facilitates subsequent detection, reduces bacterial culture steps, shortens detection time, and improves detection efficiency.

[0011] In another aspect of the invention, the present invention proposes the use of the aforementioned magnetic adsorption beads in the preparation of a kit for the detection of Staphylococcus aureus. As is known before, the binding protein in the magnetic adsorption beads can bind to Staphylococcus aureus, thereby enabling the capture and enrichment of Staphylococcus aureus by the magnetic adsorption beads for the isolation and acquisition of Staphylococcus aureus. Therefore, the kit containing the aforementioned magnetic adsorption beads can detect Staphylococcus aureus and has the advantages of short detection time and high detection efficiency.

[0012] In another aspect, the present invention provides a kit. According to an embodiment of the present invention, the kit includes the aforementioned magnetic adsorption beads. As is known before, the binding protein in the magnetic adsorption beads can bind to Staphylococcus aureus, thereby enabling the capture and enrichment of Staphylococcus aureus by the magnetic adsorption beads for the isolation and acquisition of Staphylococcus aureus. Thus, the kit containing the above-mentioned magnetic adsorption beads can detect Staphylococcus aureus and has the advantages of short detection time and high detection efficiency.

[0013] In another aspect, the present invention provides the use of the aforementioned magnetic adsorption beads or the aforementioned kit in the preparation of a product for the detection of Staphylococcus aureus. As is known, the binding protein in the magnetic adsorption beads can bind to Staphylococcus aureus, thereby enabling the capture and enrichment of Staphylococcus aureus by the magnetic adsorption beads for the isolation and acquisition of Staphylococcus aureus; and the kit containing the aforementioned magnetic adsorption beads can capture and enrich Staphylococcus aureus for the isolation and acquisition of Staphylococcus aureus. Therefore, the product of the present invention can be used to detect Staphylococcus aureus, and has the advantages of short detection time and high detection efficiency.

[0014] In another aspect, the present invention provides a method for detecting Staphylococcus aureus. According to an embodiment of the present invention, the method includes: contacting a sample to be tested with the aforementioned magnetic adsorption beads or the aforementioned reagent kit to detect whether the sample contains Staphylococcus aureus or the content of Staphylococcus aureus. As mentioned above, the aforementioned magnetic adsorption beads or reagent kit can bind to Staphylococcus aureus to achieve the capture and enrichment of the bacteria. Therefore, the method of the present invention can detect Staphylococcus aureus without the need for bacterial culture, and has the advantages of simplified detection steps, short detection time, and high detection efficiency.

[0015] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0016] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0017] Figure 1 This is a schematic diagram of different domains and cloned fragments of 80αendolysin in Embodiment 1 of the present invention;

[0018] Figure 2 This is a diagram showing the purification of 80αendolysin proteins with different domains in Example 2 of this invention;

[0019] Figure 3 This is a graph showing the results of fluorescence microscopy and flow cytometry analysis of the binding of different proteins to Staphylococcus aureus in Example 3 of the present invention.

[0020] Figure 4 This is a graph showing the results of Western spectroscopy analysis of the binding of different proteins to Staphylococcus aureus in Example 3 of the present invention;

[0021] Figure 5The results of the analysis of the stability of EGFP-amidase 3-CBD protein by different concentrations of NaCl in Example 4 of this invention;

[0022] Figure 6 The results of the analysis of the stability of EGFP-amidase 3-CBD protein at different temperatures in Example 4 of this invention;

[0023] Figure 7 The results of the analysis of the effect of different pH values ​​on the stability of EGFP-amidase 3-CBD protein in Example 4 of this invention;

[0024] Figure 8 The image shows the double enzyme digestion electrophoresis diagrams of the pET28a-EGFP-RBP recombinant plasmid in Example 5 of the present invention, wherein (A) is the double enzyme digestion electrophoresis diagram of PMV-RBP and pET28a-EGFP, and (B) is the double enzyme digestion electrophoresis diagram of pET28a-EGFP-RBP.

[0025] Figure 9 The images shown are SDS-PAGE electrophoresis diagrams of the purified EGFP-RBP protein in Example 5 of this invention, where (A) is an electrophoresis diagram of the EGFP protein and (B) is an electrophoresis diagram of the EGFP-RBP protein.

[0026] Figure 10 The image shows the binding of EGFP-RBP to Staphylococcus aureus in Example 6 of this invention, where (A) is the result of fluorescence microscopy, (B) is the result of flow cytometry quantitative analysis, and (C) is the result of Western blotting.

[0027] Figure 11 The results of fluorescence microscopy verification of the EGFP-RBP-Ni magnetic beads prepared in Example 7 of this invention;

[0028] Figure 12 The image shows the results of EGFP-amidase 3-CBD-Ni magnetic beads capturing Staphylococcus aureus in Example 7 of the present invention. (A) shows the observation results of EGFP-amidase 3-CBD-Ni magnetic beads under a fluorescence microscope, and (B) shows the observation results of EGFP-amidase 3-CBD-Ni magnetic beads under bright and dark fields under a fluorescence microscope.

[0029] Figure 13 This is a schematic diagram and recovery efficiency of EGFP-amidase 3-CBD-Ni magnetic beads in Example 7 of the present invention;

[0030] Figure 14 Sensitivity analysis of the EGFP-RBP-Ni-NTA magnetic beads in Example 7 of this invention;

[0031] Figure 15 The image shows the capture efficiency of EGFP-RBP-Ni-NTA magnetic beads in Example 7 of the present invention, where (A) is the capture efficiency of different EGFP-RBP protein concentrations and (B) is the capture efficiency of different numbers of Ni magnetic beads.

[0032] Figure 16 The analysis results of Staphylococcus aureus genomic DNA extraction by endolysin + water boiling method and water boiling method in Example 8 of this invention;

[0033] Figure 17 This is an agarose gel electrophoresis image of the NUC fragments amplified by the RPA method in Example 9 of this invention after amplification of different bacteria.

[0034] Figure 18 This is an agarose gel electrophoresis image of Staphylococcus aureus genomic DNA amplified by the RPA method at different concentrations in Example 9 of this invention;

[0035] Figure 19 This is an agarose gel electrophoresis image of Staphylococcus aureus genomic DNA amplified from samples of different sources using the RPA method in Example 10 of this invention.

[0036] Figure 20 Examples of specificity analysis (A) and sensitivity analysis (B) of EGFP-RBP-Ni magnetic bead binding PCR in Example 10 of this invention are shown.

[0037] Figure 21 Example 10 of this invention describes the detection of Staphylococcus aureus in different matrices using EGFP-RBP-Ni magnetic bead-based PCR, where lane 1 contains Tris-HCl, lane 2 contains orange juice, and lane 3 contains milk.

[0038] Figure 22 This is an agarose gel electrophoresis image of crRNA-1, crRNA-2, and crRNA-3 after purification in Example 11 of this invention;

[0039] Figure 23 This is a schematic diagram and analysis of the detection results of cas12a / crRNA cleavage in Example 11 of the present invention;

[0040] Figure 24 This study focuses on the specificity analysis of EGFP-amidase 3-CBD-Ni magnetic bead capture and cas12a / crRNA cleavage detection in Example 11 of the present invention.

[0041] Figure 25 The results of EGFP-amidase 3-CBD-Ni magnetic bead capture and binding to cas12a / crRNA cleavage detection in Example 11 of this invention are shown. Detailed Implementation

[0042] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0043] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0044] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0045] In this document, the terms “comprising” or “including” are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0046] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0047] This invention proposes an adsorption magnetic bead and its uses, a kit and its uses, and a method for detecting Staphylococcus aureus, which will be described in detail below.

[0048] Adsorption magnetic beads

[0049] In one aspect of the invention, an adsorption magnetic bead is provided. According to an embodiment of the invention, the adsorption magnetic bead comprises: Ni-polymethyl methacrylate magnetic beads; and a binding protein for binding Staphylococcus aureus, wherein the Ni-polymethyl methacrylate magnetic beads and the binding protein are linked.

[0050] The inventors also discovered that the binding protein in the magnetic beads can bind (especially specifically bind) Staphylococcus aureus. When the magnetic beads are added to the sample to be tested, the binding protein can bind Staphylococcus aureus, thus enabling the magnetic beads to capture and enrich Staphylococcus aureus. This facilitates subsequent detection, reduces the number of bacterial culture steps, shortens the detection time, and improves detection efficiency.

[0051] According to embodiments of the present invention, the above-mentioned magnetic beads may further include at least one of the following technical features:

[0052] According to an embodiment of the present invention, the N-terminus of the binding protein is connected to the Ni-polymethacrylate magnetic bead.

[0053] In this paper, the term "linked" can refer to a direct link between the binding protein and the Ni-polymethyl methacrylate (PMMA) magnetic beads, or an indirect link between the binding protein and the PMMA magnetic beads; the specific linking is not limited. For example, the binding protein and the PMMA magnetic beads can be linked via a protein tag.

[0054] According to an embodiment of the present invention, the adsorption magnetic beads further include protein tags.

[0055] In some alternative embodiments of the present invention, the Ni-polymethacrylate magnetic beads are linked to the binding protein via a His-tag sequence.

[0056] According to an embodiment of the present invention, the C-terminus of the protein tag is connected to the N-terminus of the binding protein, and the N-terminus of the protein tag is connected to the Ni-polymethacrylate magnetic bead.

[0057] According to embodiments of the present invention, the protein tag includes at least one selected from His tag, Flag tag, GST tag, MBP tag, SUMO tag and C-Myc tag.

[0058] According to an embodiment of the present invention, the protein tag is a His tag.

[0059] According to an embodiment of the present invention, the Ni-polymethacrylate magnetic beads and binding protein are provided in a manner that combines Ni and His-tag sequences.

[0060] According to embodiments of the present invention, the binding protein is derived from Staphylococcus aureus phage. The inventors further discovered that binding proteins derived from Staphylococcus aureus phage, particularly receptor-binding proteins and / or Amidase proteins derived from Staphylococcus aureus phage, can effectively achieve the capture and enrichment of Staphylococcus aureus by Ni-polymethacrylate magnetic beads, further shortening the operation time of subsequent bacterial detection steps, thereby improving detection efficiency.

[0061] In a specific embodiment, the binding protein is an endolysin of Staphylococcus aureus phage or a truncated form of the C-terminal cell wall binding domain (CBD) containing the endolysin.

[0062] In one specific embodiment, the binding protein may be Staphylococcus aureus phage lysin SA97 (LysSA97) or a truncated form of a cell wall-binding domain protein containing LysSA97. LysSA97 specifically lyses Staphylococcus aureus strains and disrupts their biofilms. It has a specific cell wall-binding domain (CBD) and two enzyme activity domains (EAD), containing a cysteine-histidine-dependent amidolytic / peptidase (CHAP, PF05257) and an N-acetylteichoyl-LL-alanine-nonamidase (amidase-3, PF01520) domain.

[0063] In one specific embodiment, the binding protein may be Staphylococcus aureus phage 80α or a truncated form containing its central amidase 3 catalytic domain (also known as Amidase 3, Amidase_3 or Amidase-3) and its C-terminal SH3b cell-binding domain (SH3b for short).

[0064] According to embodiments of the present invention, the binding protein may also be a receptor binding protein (RBP) of Staphylococcus aureus bacteriophage.

[0065] According to embodiments of the present invention, the binding protein is derived from Staphylococcus aureus phage 80α. The inventors have discovered that the protein derived from Staphylococcus aureus phage 80α has a strong binding affinity to Staphylococcus aureus. Therefore, by combining it with Ni-polymethyl methacrylate magnetic beads to prepare adsorption magnetic beads, Staphylococcus aureus in the test sample can be effectively captured, thereby improving the accuracy of Staphylococcus aureus detection in the test sample.

[0066] In some specific embodiments, the Amidase protein of the Staphylococcus aureus phage is Amidase3-CBD protein. In a more specific embodiment, the Staphylococcus aureus phage is Staphylococcus aureus phage 80α.

[0067] In this invention, the Amidase protein or Amidase 3-CBD protein specifically refers to the Amidase enzyme derived from Staphylococcus aureus bacteriophage. Amidase, also known as n-acetylmurayl-L-alanine amidase (NAMLAAEC), is a PGN hydrolase that specifically cleaves the amide bond between the lactyl group of muramic acid and the α-amino group of L-alanine. In bacteriophages, Amidases play a crucial role in the viral lysis cycle, responsible for host cell lysis, thereby allowing the release and spread of phage progeny. This invention utilizes the binding properties of Staphylococcus aureus bacteriophage Amidase protein to Staphylococcus aureus, cleverly employing the Amidase protein to achieve the specific capture and enrichment of Staphylococcus aureus by magnetic beads, thus providing a possibility for further improving and enhancing existing methods for detecting and identifying Staphylococcus aureus in food samples.

[0068] According to embodiments of the present invention, the binding protein includes at least one selected from receptor-binding proteins derived from Staphylococcus aureus phage 80α and Amidase 3CBD protein.

[0069] The inventors unexpectedly discovered that, compared with other binding proteins, receptor-binding proteins derived from Staphylococcus aureus phage 80α and Amidase 3CBD protein have a stronger binding ability to Staphylococcus aureus. Therefore, the inventors prepared adsorption magnetic beads by combining RBP and / or Amidase 3CBD protein with Ni-polymethyl methacrylate magnetic beads and used them for the detection of Staphylococcus aureus. They found that, compared with other technologies (such as antibodies), these adsorption magnetic beads have advantages such as strong stability, ligand specificity, strong affinity for carbohydrate epitopes, and high sensitivity.

[0070] According to an embodiment of the present invention, the receptor-binding protein has an amino acid sequence as shown in SEQ ID NO:13.

[0071] (SEQ ID NO:13).

[0072] Further, according to embodiments of the present invention, the receptor-binding protein includes an amino acid sequence having at least 90% homology with the amino acid sequence shown in SEQ ID NO:13 or an amino acid sequence having equivalent or similar Staphylococcus aureus binding properties to the receptor-binding protein shown in SEQ ID NO:13.

[0073] According to embodiments of the present invention, the Amidase 3-CBD protein has the amino acid sequence shown in SEQ ID NO:14. The inventors have found through experiments that the Amidase 3-CBD protein exhibits a superior capture effect on Staphylococcus aureus compared to the CBD protein.

[0074] KIMLVAGHGYNDPGAVGNGTNERDFIRKYITPNIAKYLRHAGHEVALYGGSSQSQDMYQDTAYGVNVGNKKDYGLYWVKSQGYDIVLEIHLDAAGESASGGHVIISSQFNADTIDKSIQDVIKNNLGQIRGVTPRNDLLNVNVSAEININYRLS ELGFITNKNDMDWIKKNYDLYSKLIAGAIHGKPIGGLVAGNVKTSAKNKKNPPVPAGYTLDKNNVPYKKEQGNYTVANVKGNNRRDGYSTNSRITGVLPNNTTITYDGAYCINGYRWITYIANSGQRRYIATGEVDKAGNRISSFGKFSTI(SEQ ID NO:14).

[0075] Furthermore, according to embodiments of the present invention, the Amidase 3CBD protein comprises an amino acid sequence having at least 90% homology with the amino acid sequence shown in SEQ ID NO:14 or having equivalent or similar Staphylococcus aureus binding properties to the Amidase 3CBD protein shown in SEQ ID NO:14.

[0076] According to an embodiment of the present invention, the binding protein is attached to a fluorescent group. This facilitates protein tracking.

[0077] According to an embodiment of the present invention, the fluorescent group is the EGFP group.

[0078] According to an embodiment of the present invention, the adsorption magnetic beads are prepared by the following method: the Ni-polymethacrylate magnetic beads and the binding protein are subjected to a first mixing treatment to obtain the adsorption magnetic beads.

[0079] According to an embodiment of the present invention, the binding protein is a receptor-binding protein (RBP), the amount of the receptor-binding protein added is 20-80 ng, and the amount of the Ni-polymethacrylate magnetic beads added is (1.0-10) × 10⁻⁶. 5 indivual.

[0080] In a preferred embodiment of the present invention, the amount of the receptor-binding protein added is 40-80 ng, more preferably 5-60 ng.

[0081] In a preferred embodiment of the present invention, the amount of Ni-polymethyl methacrylate magnetic beads added is (1.0~2.0)×10⁻⁶. 5 indivual.

[0082] Reagent test kit

[0083] In another aspect of the invention, a kit is provided. According to an embodiment of the invention, the kit includes the aforementioned magnetic adsorption beads. As is known before, the binding protein in the magnetic adsorption beads can bind to Staphylococcus aureus, thereby enabling the capture and enrichment of Staphylococcus aureus by the magnetic adsorption beads for the isolation and acquisition of Staphylococcus aureus. Therefore, the kit containing the aforementioned magnetic adsorption beads can detect Staphylococcus aureus and has the advantages of short detection time, high detection efficiency, and high detection sensitivity.

[0084] It should be noted that the method of providing the Ni-polymethyl methacrylate magnetic beads and the binding protein with the His-tag sequence in the kit of this invention is not strictly limited. They can be provided separately, and then brought into contact to achieve binding; or they can be provided directly as a conjugate, i.e., the Ni-polymethyl methacrylate magnetic beads and the binding protein are bound together by the Ni and His-tag sequences, i.e., provided in the form of the aforementioned adsorption magnetic beads. When in use, this conjugate is directly brought into contact with the sample to be tested to achieve the purpose of capturing pathogens.

[0085] According to an embodiment of the present invention, the kit further comprises at least one of Cas12a, crRNA, and ssDNA-FQreporter.

[0086] According to embodiments of the present invention, the kit further comprises Cas12a, crRNA, and ssDNA-FQ reporter. The Cas12a protein binds to crRNA to form a complex, which then binds to the amplification product. When the complex recognizes the target sequence in the amplification product, the DNase region in Cas12a is activated, first cleaving the target sequence fragment, then cleaving the single-stranded ssDNA, and generating a detectable fluorescent signal. Therefore, by mixing and reacting the amplification product, Cas12a, crRNA, and ssDNA-FQ reporter at room temperature (30-40℃), the DNA fragment in the amplification product can be rapidly detected without the need for instruments, making the operation simple.

[0087] use

[0088] In another aspect, the present invention proposes the use of the aforementioned magnetic adsorption beads in the preparation of a kit for the detection of Staphylococcus aureus. As is known prior, the binding protein in the magnetic adsorption beads can bind to Staphylococcus aureus, thereby enabling the capture and enrichment of Staphylococcus aureus by the magnetic adsorption beads for the isolation and acquisition of Staphylococcus aureus. Therefore, the kit containing the aforementioned magnetic adsorption beads can detect Staphylococcus aureus and has advantages such as short detection time, high detection efficiency, and high detection sensitivity.

[0089] In another aspect, the present invention provides the use of the aforementioned magnetic adsorption beads or the aforementioned kit in the preparation of a product for the detection of Staphylococcus aureus. As is known, the binding protein in the magnetic adsorption beads can bind to Staphylococcus aureus, thereby enabling the capture and enrichment of Staphylococcus aureus by the magnetic adsorption beads for the isolation and acquisition of Staphylococcus aureus; and the kit containing the aforementioned magnetic adsorption beads can capture and enrich Staphylococcus aureus for the isolation and acquisition of Staphylococcus aureus. Therefore, the product of the present invention can be used to detect Staphylococcus aureus, and has the advantages of short detection time, high detection efficiency, and high detection sensitivity.

[0090] Methods for detecting Staphylococcus aureus

[0091] In another aspect, the present invention provides a method for detecting Staphylococcus aureus. According to an embodiment of the present invention, the method includes: contacting a sample to be tested with the aforementioned magnetic adsorption beads or the aforementioned reagent kit to detect whether the sample contains Staphylococcus aureus or the content of Staphylococcus aureus. As mentioned above, the aforementioned magnetic adsorption beads or reagent kit can bind to Staphylococcus aureus to achieve the capture and enrichment of the bacteria. Therefore, the method of the present invention can detect Staphylococcus aureus without the need for bacterial culture, and has the advantages of simplified detection steps, short detection time, high detection efficiency, and high detection sensitivity.

[0092] It should be noted that the method of the present invention is a non-disease diagnostic method, used to detect Staphylococcus aureus in food, articles, and other non-animal tissue or blood samples.

[0093] According to an embodiment of the present invention, the method further includes: performing DNA extraction treatment on the contact treatment product; performing amplification treatment on the DNA extraction product; and detecting the amplification product to detect whether the sample to be tested contains Staphylococcus aureus or the content of Staphylococcus aureus.

[0094] According to an embodiment of the present invention, in one specific implementation, the adsorption magnetic beads are obtained by first mixing Ni-polymethyl methacrylate magnetic beads and binding protein.

[0095] According to an embodiment of the present invention, the binding protein is Amidase 3-CBD protein, and the first mixing treatment is carried out at a temperature of (2-30) °C and a pH of 5-9. This improves the capture efficiency of the magnetic beads for Staphylococcus aureus.

[0096] According to an embodiment of the present invention, the binding protein is Amidase 3-CBD protein, and NaCl and the adsorbed magnetic beads are subjected to a second mixing treatment before the contact treatment.

[0097] According to embodiments of the present invention, the final concentration of NaCl is (50-200) mM, for example (50-150) mM, (50-100) mM, preferably 100 mM. This results in better binding of the binding protein on the magnetic beads to Staphylococcus aureus. In particular, as the NaCl concentration increases, the binding effect of Amidase 3-CBD protein to Staphylococcus aureus continuously weakens.

[0098] According to an embodiment of the present invention, the number of adsorption magnetic beads added is not less than 1.0 × 10⁻⁶. 5 For example, not less than 1.1 × 10 5 1.2 × 10 5 1.3 × 10 5 1.4 × 10 5 1.5 × 10 5 1, not less than 2.0 × 10 5 1, not less than 1.0 × 10 6 1, not less than 1.0 × 10 7 This can further enhance the capture ability of the binding protein of the magnetic beads for Staphylococcus aureus, especially the capture ability of RBP for Staphylococcus aureus.

[0099] According to an embodiment of the present invention, the DNA extraction process includes: boiling the contact treatment product with an endolysin in water. Through extensive experimentation, the inventors discovered that an endolysin can specifically lyse the cell wall of Staphylococcus aureus, leading to the release of intracellular substances. DNA from Staphylococcus aureus can then be extracted by boiling in water, significantly increasing the extraction yield. This method offers advantages such as shorter extraction time, ease of operation, and high extraction volume.

[0100] According to an embodiment of the present invention, before the boiling treatment, the endosomalin and the contact treatment product are reacted for 20-40 minutes. This allows for thorough lysis of the Staphylococcus aureus cell wall, increasing the extraction yield of Staphylococcus aureus DNA.

[0101] According to an embodiment of the present invention, the boiling treatment time is 5-20 minutes. Therefore, the extraction effect of Staphylococcus aureus DNA is better.

[0102] According to an embodiment of the present invention, the volume ratio of the endolysin to the sample to be tested is 1:(40-60), wherein the concentration of the endolysin is (1-3) mg / ml. Therefore, the extraction effect of Staphylococcus aureus DNA is better.

[0103] It should be noted that the sample to be tested can be pretreated before being processed with the above-mentioned magnetic beads or the above-mentioned kit. Pretreatment includes, but is not limited to, dilution and impurity removal. The specific method is not limited.

[0104] According to an embodiment of the present invention, the endolysin is selected from bacteriophage endolysins derived from the Staphylococcus aureus.

[0105] According to an embodiment of the present invention, the endolysin is selected from phage 80α endolysin (abbreviated as endolysin or 80α endolysin) derived from the Staphylococcus aureus.

[0106] According to an embodiment of the present invention, the phage 80α endolysin has an amino acid sequence as shown in SEQ ID NO:15.

[0107] MLMTKNQAEKWFDNSLGKQFNPDGWYGFQCYDYANMFFMLATGERLQGLYAYNIPFDNKAKIEKYGQIIKNYDSFLPQKLDIVVFPSKYGGGAGHVEIVESANLNTFTSFGQNWNGKGWTNG VAQPGWGPETVTRHVHYYDNPMYFIRLNFPNNLSVGNKAKGIIKQATTKKEAVIKPKKIMLVAGHGYNDPGAVGNGTNERDFIRKYITPNIAKYLRHAGHEVALYGGSSQSQDMYQDTAYGV NVGNKKDYGLYWVKSQGYDIVLEIHLDAAGESASGGHVIISSQFNADTIDKSIQDVIKNNLGQIRGVTPRNDLLNVNVSAEININYRLSELGFITNKNDMDWIKKNYDLYSKLIAGAIHGKP IGGLVAGNVKTSAKNKKNPPVPAGYTLDKNNVPYKKEQGNYTVANVKGNNRDGYSTNSRITGVLPNNTTITYDGAYCINGYRWITYIANSGQRRYIATGEVDKAGNRISSFGKFSTI(SEQ ID NO:15).

[0108] It should be noted that the endosomalin was obtained using conventional cloning and expression methods.

[0109] According to an embodiment of the present invention, the amplification process is performed using a recombinase polymerase amplification method. Therefore, the recombinase polymerase amplification (RPA) method can operate at a low-temperature isothermal environment, does not require template thermal denaturation, and does not require instruments, offering advantages such as simple operation. Specifically, the present invention does not strictly limit the specific steps and required reagents of the recombinase polymerase amplification method, and these can be obtained using conventional methods in the art.

[0110] According to an embodiment of the present invention, the detection includes: performing a third mixing treatment on the amplification product, Cas12a, crRNA, and ssDNA-FQ reporter; and detecting whether the sample contains Staphylococcus aureus or the content of Staphylococcus aureus based on the fluorescence signal generated by the third mixing treatment. Specifically, after the Cas12a protein binds to crRNA to form a complex, it binds to the amplification product. When the complex recognizes the target sequence in the amplification product, the DNase region in Cas12a is activated, first cleaving the target sequence fragment, then cleaving the single-stranded ssDNA, and generating a detectable fluorescence signal. Therefore, under room temperature (30-40℃) conditions, mixing and reacting the amplification product, Cas12a, crRNA, and ssDNA-FQ reporter allows for rapid detection of DNA fragments in the amplification product without the need for instruments, making the operation simple.

[0111] According to an embodiment of the present invention, the third mixing treatment generates a fluorescent signal, which is an indication that the sample to be tested contains Staphylococcus aureus; or, the third mixing treatment does not generate a fluorescent signal, which is an indication that the sample to be tested does not contain Staphylococcus aureus.

[0112] According to an embodiment of the present invention, the detection further includes: determining the content of Staphylococcus aureus in the sample to be tested based on a standard curve, wherein the standard curve is a curve corresponding to a predetermined amount of Staphylococcus aureus and the intensity of a fluorescence signal.

[0113] According to embodiments of the present invention, the crRNA has a nucleotide sequence as shown in SEQ ID NO:7 or a nucleotide sequence having at least 90% homology with it. This further improves the sensitivity for detecting Staphylococcus aureus, with a detection limit of 1 × 10⁻⁶. 1 CFU / mL.

[0114] UAAUACGACUCACUAUAGGGUAAUUUCUACUAAGUGUAGAUGUUGAAGUUGCACUAUAUAC (SEQ ID NO: 7).

[0115] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0116] The nucleotide sequences in the following examples can be obtained from their corresponding amino acid sequences using conventional methods or conventional software (e.g., online programs like Vectorbuilder (https: / / www.vectorbuilder.cn / tool / codon-optimization.html), GeneOptimizer, etc.).

[0117] Example 1: Bioinformatics Analysis of Staphylococcus aureus Bacteriophage 80α Endolysin

[0118] The 80α Endolysin gene was identified from the phage 80α genome. The 80α Endolysin domain was analyzed using Interpro, and the molecular weight and isoelectric point of the protein were calculated using the pI / Mw calculation program ExPASy.

[0119] Analysis of the phage 80α genome revealed the 80α Endolysin gene sequence. Interpro was used to search for functional domains, showing that the 80α Endolysin gene consists of three domains: the N-terminal CHAP catalytic domain (CHAP), the central amidase 3 catalytic domain (also known as Amidase 3, Amidase_3, or Amidase-3), and the C-terminal SH3b cell-binding domain (SH3b). Figure 1 As shown.

[0120] Therefore, the present invention subsequently selected the Amidase 3-CBD and CBD gene segments for cloning and expression. The amino acid sequence of phage 80α Endolysin is shown in SEQ ID NO:15, the amino acid sequence of Amidase 3-CBD is shown in SEQ ID NO:14, and the amino acid sequence of CBD is shown in SEQ ID NO:16.

[0121] YKKEQGNYTVANVKGNVRDGYSTNSRITGVLPNNTTITYDGAYCINGYRWITYIANSGQRRYIATGEVDKAGNRIS SFGKFSTI (SEQ ID NO: 16).

[0122] Example 2: Cloning, expression, and purification of EGFP-Amidase 3-CBD and EGFP-CBD proteins

[0123] The nucleotide sequences corresponding to the Amidase 3-CBD and CBD proteins in Example 1 were cloned into the PET28a-EGFP vector. The recombinant proteins were obtained by purification using the E. coli gene expression system and Ni-NTA affinity chromatography column. Amidase 3-CBD and CBD with EGFP were obtained, respectively, and named EGFP-Amidase 3-CBD and EGFP-CBD. SDS-PAGE analysis was performed.

[0124] like Figure 2 As shown, the results indicate that the size of the EGFP-Amidase 3-CBD protein is approximately 60 kDa (i.e., it is approximately 60 kDa). Figure 2 The purified product from lane 8 of swim grout A), the EGFP-CBD protein is approximately 40 kDa in size. Figure 2 The purified products from lane 7 of pool B, and the protein sizes of both on SDS-PAGE gels matched the bioinformatics predictions of 64 and 40.1 kDa, respectively. Figure 2 A and Figure 2 B), and simultaneously, the protein size of the negative control EGFP protein on the SDS-PAGE gel (approximately 33 kDa) matched the bioinformatics prediction (33.2 kDa). Figure 2 (Purified product of lane 8 in C).

[0125] Example 3: Analysis of EGFP-Amidase 3-CBD and EGFP-CBD binding activity

[0126] Experimental method: Staphylococcus aureus was grown to mid-log phase under shaking culture at 37℃. The culture was then centrifuged at 12000 rpm for 5 min, washed once with 50 mM Tris-HCl, centrifuged again, and the bacteria were resuspended in 50 mM Tris-HCl. The OD values ​​were then recorded. 600 Adjust to 0.8. Take 200 μl of OD. 600The bacterial culture was placed in centrifuge tubes at a concentration of 0.8 μL. 100 μL of 0.5 mg / mL EGFP-Amidase 3-CBD and EGFP-CBD proteins obtained in Example 2 were added, and the tubes were incubated at room temperature for 20 minutes. Staphylococcus aureus was washed three times with 50 mM Tris-HCl by centrifugation (10000 rpm, 3 min) to remove unbound proteins. The washed precipitate was resuspended in 100 μL of 50 mM Tris-HCl to obtain a bacterial protein suspension, which was then analyzed using fluorescence microscopy, flow cytometry, and Western blotting.

[0127] Fluorescence microscopy detection: 10 μl of the final bacterial protein suspension was added to a glass slide, and a coverslip was placed on top. The binding effect of the protein with Staphylococcus aureus was observed under a bright field and FITC filter using an epifluorescence microscope equipped with a U-RFL-T light source (1000x magnification). Excitation: BP 470-490 nm; Emission: LP 516 nm. EGFP protein was prepared as a negative control. To evaluate the specificity and sensitivity of EGFP-Amidase 3-CBD, bacteria listed in the following categories—Staphylococcus aureus, Escherichia coli O157:H, Salmonella typHimurium, Bacillus cereus, Propionibacterium acnes, and Listeria monocytogenes—were incubated with the protein (using the same procedures as described above). The results were observed under an epifluorescence microscope following the same procedure. The results are shown in Table 1.

[0128] Table 1: The lysis of cell walls of different microorganisms by 80αendolysin and EGFP-amidase 3-CBD

[0129]

[0130]

[0131] Note: a + indicates cleavage activity; - indicates no cleavage activity. b + indicates cell wall binding activity; - indicates no cell wall binding activity.

[0132] Flow cytometry analysis: 50 μl of the final bacterial protein suspension was analyzed using an Accuri C6 Plus flow cytometer equipped with a diode blue laser (excitation wavelength 488 nm), collecting 40,000 events. Fluorescence was detected using a 525 / 50 nm bandpass filter on the FL1 channel, and data analysis was performed using FlowJo-V10 software.

[0133] Western blot analysis: 5 μl of 5×SDS loading buffer was added to 20 μl of the final bacterial protein suspension, boiled at 100°C for 5 minutes, and analyzed by electrophoresis using an SDS-PAGE pre-prepared gel. The protein was then transferred to a PVDF membrane using a transfer technique and analyzed by Western blot analysis using an antihistamine antibody.

[0134] Fluorescence microscopy results showed that both the fusion proteins EGFP-Amidase 3-CBD and EGFP-CBD could bind to Staphylococcus aureus cells; however, fluorescence intensity observation under the microscope indicated that EGFP-Amidase 3-CBD had a stronger binding ability than EGFP-CBD. No fluorescent cells were observed in the negative control EGFP under the fluorescence microscope. (See details...) Figure 3 A.

[0135] like Figure 3 B and Figure 3 As shown in Figure C, flow cytometry results revealed a significant rightward shift in the peak value of EGFP-Amidase 3-CBD compared to the positive control, and the average fluorescence intensity of EGFP-Amidase 3-CBD was significantly higher than that of EGFP-CBD (P<0.0001). This indicates that EGFP-Amidase 3-CBD possesses a stronger binding affinity than EGFP-CBD. Figure 3 In C, the negative control EGFP also showed an average fluorescence intensity value. The analysis showed that this was due to the failure to wash away the unbound protein after it bound to Staphylococcus aureus, which is a normal experimental error.

[0136] like Figure 4 As shown, the gray value of the EGFP-Amidase 3-CBD band is higher than that of EGFP-CBD. Furthermore, the comparison of non-specific bands also shows that the non-specific band of EGFP-Amidase 3-CBD is much lighter than that of EGFP-CBD, indicating that EGFP-Amidase 3-CBD has a stronger binding ability than EGFP-CBD.

[0137] Example 4: Stability assessment of EGFP-amidase 3-CBD protein

[0138] The stability of the EGFP-amidase 3-CBD protein obtained in Example 2 was investigated at temperatures ranging from 4 to 60°C, pH values ​​ranging from 3 to 12, and NaCl concentrations ranging from 0 to 1000 mM. The protein was treated for 30 min under each condition. The binding activity of amidase 3-CBD to Staphylococcus aureus was determined using the functional assay method of 80α Endolysin C-terminus.

[0139] The different pH conditions were achieved using buffer solutions of different pH values: glycine-HCl pH=3, sodium acetate pH=5, Tris-HCl pH=7, glycine-NaOH pH=9, and KCl-NaOH pH=12.

[0140] Under each condition, the protein treatment time was 30 min, and the binding activity of amidase 3-SH3b to bacteria was determined using the functional assay method of 80α Endolysin C-terminus.

[0141] like Figure 5 As shown, the results indicate that the binding ability of EGFP-amidase 3-CBD protein to Staphylococcus aureus is strongest after treatment with 100 mM NaCl. With increasing NaCl concentration, the binding ability of EGFP-amidase 3-CBD protein to Staphylococcus aureus gradually weakens. In lyophilization assays, the EGFP-amidase 3-CBD protein treated with 100 mM NaCl exhibits the highest fluorescence peak and average fluorescence intensity. When the NaCl concentration exceeds 200 mM, the binding ability of EGFP-amidase 3-CBD significantly decreases.

[0142] like Figure 6-7 As shown in the results of treating EGFP-amidase 3-CBD protein at different temperatures and pH values, the EGFP-amidase 3-CBD protein exhibits the best binding effect on Staphylococcus aureus when the temperature is 4℃ and the pH is 7.

[0143] Example 5: Expression and purification of EGFP-RBP

[0144] 1. The phage 80αRBP gene sequence was obtained from NCBI (Genbank accession number ABF71632.1), and simulated cloning was performed using snapgene software. The molecular weight and isoelectric point of the protein were calculated using ExPASy. The amino acid sequence of phage 80αRBP is shown in SEQ ID NO:13.

[0145] 2. The gene encoding phage 80αRBP from step 1 was artificially synthesized, and the synthesized gene sequence (DNA sequence) was cloned into the PMV vector to obtain the plasmid PMV-RBP. It was then cloned into the pET28 vector containing EGFP using double enzyme digestion (BamHI / XhoI) to generate the pET28a-EGFP-RBP recombinant expression vector. The recombinant expression vector was transformed into *E. coli* DH5α competent cells using the heat shock method. Single colonies were then picked and cultured overnight at 37°C in LB broth containing kanamycin. The plasmid was then extracted using the alkaline lysis method and sequenced (sequencing results are shown in the figure). Figure 8 As shown in the figure, the sequence with the correct sequence is identified as a positive transformant.

[0146] 3. Transform the correctly sequenced pET28a-EGFP-RBP from step 2 into Escherichia coli BL21(DE3) strain, and then... 600 When the concentration reached approximately 0.6, IPTG (isopropyl-β-d-thiogalactoside) was added to the culture to a final concentration of 1 mM, and protein expression was induced at 19°C for 20 h. For purification, the harvested cells were resuspended in lysis buffer (50 mM Tris-HCl, 500 mM NaCl, 5 mM imidazole), sonicated for 3 min, and then centrifuged at 10,000 rpm for 30 min to obtain soluble protein supernatant. The collected supernatant sample was added to a pre-equilibrated Ni column, and the solution flowing out of the Ni column was used as the permeation buffer. The target protein in the permeation buffer was then eluted sequentially using a buffer containing 50 mM imidazole (washing solution), a buffer containing 250 mM imidazole (washing solution 1), and an elution buffer containing 500 mM imidazole (washing solution 2). After this, the protein was concentrated using a concentration column and the buffer was replaced. The protein was then stored in 50 mM Tris-HCl. After purification, proteins were analyzed using a pre-prepared gel for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein concentrations were determined using a Bradford protein assay kit; results are shown below. Figure 9 .

[0147] Figure 9 A shows that the protein size (approximately 33 kDa) of the negative control EGFP protein on an SDS-PAGE gel matches the bioinformatics-predicted molecular weight (33.2 kDa). Figure 9 B shows that the purified product, analyzed by SDS-PAGE electrophoresis, showed a single band of approximately 100 kDa, which matches the molecular weight predicted by bioinformatics (104.1 kDa).

[0148] The above experimental results show that phage 80αRBP was successfully expressed and purified.

[0149] Example 6: Investigation of the binding activity of EGFP-RBP with Staphylococcus aureus

[0150] The binding ability of EGFP-RBP obtained in Example 5 to Staphylococcus aureus was detected by fluorescence microscopy, flow cytometry, and Western blotting.

[0151] Experimental method: Staphylococcus aureus was cultured in LB broth at 37℃ to the logarithmic phase, then the culture was centrifuged at 12000 rpm for 5 min, and then resuspended in 50 mM Tris-HCl. 600 Adjust to 0.6. Take 100 μL of OD. 600 A bacterial culture with a pH of 0.6 was placed in a centrifuge tube, and 50 μL of 0.5 mg / mL EGFP-RBP protein was added. The tubes were incubated at room temperature for 30 min. The bacteria were then washed three times with 50 mM Tris-HCl by centrifugation (10000 rpm, 3 min) to remove unbound protein. The washed precipitate was resuspended in 100 μL of 50 mM Tris-HCl.

[0152] Fluorescence microscopy: 10 μL of resuspension was added to a glass slide and covered with a coverslip. The binding effect of the protein with bacteria was observed under bright field and FITC filters (excitation BP 470-490 nm; emission LP 516 nm). Bacterial cells supplemented with EGFP protein were prepared as a negative control. When 80αRBP was fused with green fluorescent protein (EGFP) for expression, the binding ability of RBP to Staphylococcus aureus could be directly observed through fluorescence imaging.

[0153] Flow cytometry analysis: 50 μL of resuspended bacterial culture was used to analyze the sample using an Accuri C6 Plus flow cytometer equipped with a diode blue laser (excitation wavelength of 488 nm). A total of 10,000 events were collected, and fluorescence was detected using a 533 / 30 nm fluorescence detector on the FL1 channel. Data analysis was performed using FlowJo-V10 software.

[0154] Western Blot assay: 20 μL of resuspended bacterial culture was mixed with 5 μL of 5×SDS loading buffer and boiled at 100°C for 5 minutes. Electrophoresis was performed using a pre-prepared SDS-PAGE gel. Proteins were then transferred to a PVDF membrane using a transfer technique. Western blot analysis was performed using a primary antihistamine antibody and an HRP-labeled secondary antibody. Protein bands were visualized using an ECL kit and an image analyzer.

[0155] Fluorescence microscopy results showed that ( Figure 10A) EGFP-RBP can bind to Staphylococcus aureus cells and exhibit green fluorescence under blue excitation light.

[0156] Flow cytometry results showed ( Figure 10 B) The average fluorescence intensity of EGFP-RBP was significantly stronger than that of EGFP, and the two showed a highly significant difference (P<0.0001).

[0157] Western blotting results also showed ( Figure 10 C) The gray value of the EGFP-RBP band is higher than that of EGFP.

[0158] The results of the three experiments above all confirmed that EGFP-RBP has the ability to bind to Staphylococcus aureus.

[0159] Example 7: Investigation on the efficiency of Ni-NTA magnetic beads with binding protein in capturing Staphylococcus aureus

[0160] 1. Preparation of EGFP-amidase 3-CBD-Ni-NTA magnetic beads (also known as EGFP-amidase 3-CBD-Ni magnetic beads)

[0161] Ni-polymethyl methacrylate magnetic beads (Ni-NTA magnetic beads, Wuxi Baimaige Biotechnology Co., Ltd.) have Ni-polymethyl methacrylate groups on their surface. The Ni ions, which are magnetic affinity ligands on the Ni-polymethyl methacrylate, can bind to the N-terminal 6×His-tag tag on the protein. In Example 2, the N-terminus of the EGFP-amidase 3-CBD protein was fused with a 6×His-tag tag. Then, 100 μl of EGFP-amidase 3-CBD protein containing the His-tag tag (buffer formulation: 50 mM tris-HCl, 100 mM NaCl, pH = 7.4) was incubated with 100 μl of Ni-polymethyl methacrylate magnetic beads and mixed in a mixer for 20 min. The EGFP-amidase 3-CBD protein was added in excess compared to the Ni-polymethyl methacrylate magnetic beads. The Ni-polymethyl methacrylate magnetic beads were then separated using a magnetic rack and washed twice with T100. The beads were washed twice with 50 mM Tris-HCl, and then resuspended in 100 μl of 50 mM Tris-HCl to form a magnetic bead suspension. The suspension was stored at 4 °C, and the coating of Ni-polymethyl methacrylate magnetic beads was observed using a fluorescence microscope. The results showed that no fluorescence signal was observed in the magnetic beads that were not labeled with EGFP-amidase 3-CBD protein under a FITC filter, while a strong green fluorescence signal was observed in the magnetic beads labeled with EGFP-amidase 3-CBD protein under a FITC filter.

[0162] 2. Preparation of EGFP-RBP-Ni-NTA magnetic beads (also known as EGFP-RBP-Ni magnetic beads)

[0163] (1) Take 100 μL of Ni-polymethyl methacrylate magnetic beads (Ni-NTA magnetic beads, Wuxi Baimaige Biotechnology Co., Ltd.) and place them in a 1.5 mL EP tube. Place the tube in a magnetic rack, discard the supernatant, add 500 μL of 50 mM Tris-HCl to resuspend the magnetic beads, mix well, place the tube in a magnetic separator, discard the supernatant, wash the tube repeatedly 3 times, and finally suspend the tube in 100 μL of 50 mM Tris-HCl.

[0164] (2) Add 60 μL of EGFP-RBP (1 mg / mL) protein prepared in Example 5. The N-terminus of the EGFP-RBP protein contains a 6×His-tag. Manually rotate and mix for 20 min to label the magnetic beads, then discard the supernatant.

[0165] (3) Add 500 μL of 50 mM Tris-HCl to resuspend the magnetic beads, mix well, place on a magnetic separator for 10 s, discard the supernatant, wash twice, and finally suspend in 100 μL of 50 mM Tris-HCl to obtain the magnetic bead suspension. Take 10 μL of the sample and observe it with a fluorescence microscope. Store the remainder at -4℃.

[0166] from Figure 11 The results showed that no fluorescence signal was observed in the magnetic beads unlabeled with EGFP-RBP protein under a FITC filter, while a strong green fluorescence signal was observed in the magnetic beads labeled with EGFP-RBP protein under a FITC filter. Furthermore, through fluorescence microscopy, the inventors were surprised to observe that the EGFP-RBP protein was uniformly covered on the surface of the Ni-NTA magnetic beads. After adding EGFP-RBP, it was found that the green fluorescence observed at an excitation peak of 488 nm corresponded one-to-one with the position of Staphylococcus aureus in the bright field.

[0167] The above results indicate that the EGFP-RBP protein obtained in this invention can be easily uniformly coated on the surface of Ni-polymethacrylate magnetic beads, and the EGFP-RBP-Ni magnetic beads (also known as EGFP-RBP-Ni-NTA magnetic beads) were successfully prepared.

[0168] 3. The Ni-polymethyl methacrylate magnetic beads coated with EGFP-RBP protein (EGFP-RBP-Ni-NTA magnetic beads) and the Ni-polymethyl methacrylate magnetic beads coated with EGFP-amidase 3-CBD protein (EGFP-amidase 3-CBD-Ni-NTA magnetic beads) obtained above were used to detect the capture efficiency of Staphylococcus aureus. The magnetic beads that captured Staphylococcus aureus were plate-cultured and counted. The specific operation is as follows:

[0169] Add 1 ml of Staphylococcus aureus (10 1 -10 4 Incubate 100 μl of EGFP-amidase 3-CBD-Ni-NTA magnetic bead suspension with a CFU / ml solution on a mixer for 40 min. Separate the magnetic beads using a magnetic rack, wash twice with 50 mM Tris-HCl, and finally resuspend the magnetic beads in 1 ml of 50 mM Tris-HCl. Spread the 1 ml magnetic bead suspension directly onto a plate and incubate at 37°C for 24 h. After colony counting, the results for EGFP-amidase 3-CBD-Ni-NTA magnetic beads are as follows: Figures 12-13 For the results of EGFP-RBP-Ni-NTA magnetic beads, please refer to [link / reference]. Figure 14 The magnetic bead capture efficiency (100%) is calculated as follows: (Number of magnetic beads captured / Number of supernatant residues + Number of magnetic beads captured) × 100%.

[0170] like Figure 12 As shown, observation under a fluorescence microscope revealed that the EGFP-amidase 3-CBD protein was very uniformly coated on the surface of Ni-polymethyl methacrylate magnetic beads. Furthermore, after adding RFP-amidase 3-CBD, observations at excitation peaks of 488 nm and 532 nm showed that the positions of the observed green and red fluorescence and the bright-field Staphylococcus aureus were identical. These results demonstrate that the EGFP-amidase 3-CBD protein obtained in this invention can easily achieve uniform coating on the surface of Ni-polymethyl methacrylate magnetic beads.

[0171] like Figure 13 As shown, Ni-polymethyl methacrylate magnetic beads coated with EGFP-amidase 3-CBD protein were used in Staphylococcus aureus bioburden at 1×10⁻⁶. 4 The capture efficiency was 25% at CFU / ml; as the bacterial load gradually decreased, the capture efficiency of the magnetic beads also increased, reaching 1×10⁻⁶. 1 At a concentration of CFU / ml, the magnetic beads achieve a capture efficiency of 78%, resulting in excellent capture performance.

[0172] like Figure 14 As shown, when the number of Staphylococcus aureus is 1×104 At CFU (Continuous Fusion), the number of EGFP-RBP-Ni-NTA magnetic beads is insufficient to capture all Staphylococcus aureus. However, as the number of Staphylococcus aureus decreases, the capture efficiency of the magnetic beads continuously increases, reaching 1×10⁻⁶. 1 At CFU / ml, the number of magnetic beads is sufficient relative to the number of bacteria, and the capture efficiency of the magnetic beads reaches 82%, thus increasing the efficiency of recovering Staphylococcus aureus.

[0173] Furthermore, the inventors optimized the capture conditions of Staphylococcus aureus by the prepared EGFP-RBP-Ni-NTA magnetic beads.

[0174] 4. Optimization of EGFP-RBP-Ni-NTA magnetic bead capture conditions

[0175] (1) EGFP-RBP protein concentration

[0176] The concentration of EGFP-RBP protein directly affects the labeling efficiency of magnetic beads, and thus the capture rate of target bacteria. In this experiment, magnetic beads were labeled with EGFP-RBP protein concentrations of 10 ng, 60 ng, and 100 ng, respectively, and Staphylococcus aureus was serially diluted with 50 mM Tris-HCl to approximately 1 × 10⁻⁶. 3 CFU / mL. Follow step 3 to perform the experiment, calculate the magnetic bead capture efficiency, and then determine the optimal EGFP-RBP protein concentration.

[0177] Depend on Figure 15 As shown in Figure A, when the amount of EGFP-RBP protein added was 10 ng labeled magnetic beads, the capture efficiency of EGFP-RBP-Ni-NTA magnetic beads was 50%. With the increase of the amount of EGFP-RBP protein added, the recovery rate of the magnetic beads also increased accordingly. However, when the amount of protein added reached 100 ng, the recovery rate of the magnetic beads was 65%, which showed no significant difference in capture rate compared to the amount of protein added at 60 ng (P>0.05). This may be because the amount of EGFP-RBP protein added at 60 ng had already saturated the magnetic beads. Therefore, an addition amount of 60 ng of EGFP-RBP protein was chosen for the next experimental step.

[0178] (2) Number of Ni-NTA magnetic beads

[0179] In this experiment, Staphylococcus aureus was diluted to approximately 1×10⁻⁶ using 50 mM Tris-HCl. 3 CFU / mL, select to add 6×10 4 7.2×10 4 9×10 4 1.2×10 5Coupling and capture experiments were conducted with different numbers of EGFP-RBP-Ni-NTA magnetic beads (see step 3 of this embodiment for specific steps). The capture rate was calculated based on the number of colonies on the plate to determine the optimal number of Ni-polymethyl methacrylate magnetic beads.

[0180] Depend on Figure 15 As shown in Figure B, with the continuous increase of the number of Ni-polymethyl methacrylate magnetic beads, the capture rate of the magnetic beads also increases accordingly, reaching 1.2 × 10⁻⁶. 5 At a specific number of magnetic beads, the recovery rate reached 60%. However, this correlation is not always maintained. Too many magnetic beads may reduce the capture efficiency of the target bacteria, and effective dispersion may be difficult during capture. Therefore, for EGFP-RBP-Ni-NTA magnetic beads, a concentration of 1.2 × 10⁻⁶ was selected. 5 The following experiment was conducted with a number of magnetic beads.

[0181] Example 8: Investigation of Staphylococcus aureus genome extraction using different extraction methods

[0182] 1. The steps for extracting Staphylococcus aureus genomic DNA using the endolysin + water boiling method are as follows:

[0183] The 80α Endolysin gene was identified from the phage 80α genome, synthesized by BGI Genomics, and cloned into the PMV vector. The 80α Endolysin gene was then cloned into the PET28a vector via double enzyme digestion (BamHI / XhoI). The constructed PET28a-endolysin was transformed into BL21 cells and processed at OD... 600 Add 1 mM IPTG when the concentration of α=0.6 and induce at 19°C for 20 h. Collect the induced bacteria at 10000 RPM for 5 min, then resuspend the bacteria in lysis buffer (50 mM Tris-HCl, 500 mM NaCl, 5 mM imidazole), sonicate for 3 min, and centrifuge at 10000 rpm for 30 min to obtain soluble protein supernatant. Then, purify the target protein using EGFP-amidase 3-CBD-Ni-NTA magnetic beads obtained in Example 2. After that, concentrate the protein using Amicon Ultra-4 centrifugal filters Ultracel-30K and replace the elution buffer (50 mM Tris-HCl, 500 mM imidazole, 100 mM NaCl). Store the protein (the amino acid sequence of 80α Endolysin protein is shown in SEQ ID NO:15) in 50 mM Tris-HCl. After purification, analyze the protein using a pre-prepared gel for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Figure 16As shown in Figure A, the purified 80αendolysin protein, as analyzed by SDS-PAGE, has a size of approximately 57 kDa (i.e., ). Figure 16 The purified product from lane 9 of pool A matched the bioinformatics predicted size of 80α endolysin protein (57.3 kDa). Therefore, 80α Endolysin was obtained.

[0184] Take 1 mL OD 600 Staphylococcus aureus bacterial suspension (Staphylococcus aureus ATCC29213) at pH 0.8 was placed in a centrifuge tube and centrifuged at 10,000 rpm for 3 min, and the supernatant was discarded; 100 μl of Tris-HCl (pH = 7.4) buffer was added to suspend the bacteria; 40 μl of 400 μg / ml 80αendolysin protein obtained above was added, and the reaction was carried out for 30 min; the mixture was then boiled in a water bath for 10 min, centrifuged at 10,000 rpm for 3 min, and the supernatant was collected for qPCR.

[0185] The qPCR system is shown below:

[0186] Sybergreen mix 5μl Nuc(F):5'-GGCATATGTATGGCAATTGTTTC-3'(SQE ID NO:1) 0.3μl Nuc(R):5'-CGTATTGCCCTTTCGAAACATT-3'(SQE ID NO:2) 0.3μl <![CDATA[H2O]]> 3.4μl

[0187] The cycle system was: 95℃, 15min; 95℃, 15s; 60℃, 20s; 30 cycles; 72℃, 45s.

[0188] 2. The steps for extracting Staphylococcus aureus genomic DNA using the boiling method are as follows:

[0189] Take 1 mL OD 600 Staphylococcus aureus culture (Staphylococcus aureus ATCC29213) at pH 0.8 was placed in a centrifuge tube and centrifuged at 10,000 rpm for 3 min. The supernatant was discarded. 140 μl of Tris-HCl (pH 7.4) buffer was added to suspend the bacteria. The mixture was then incubated in a 100°C water bath for 10 min, centrifuged at 10,000 rpm for 3 min, and the supernatant was collected for qPCR.

[0190] The qPCR system is shown below:

[0191] Sybergreen mix 5μl Nuc(F):5'-GGCATATGTATGGCAATTGTTTC-3'(SQE ID NO:1) 0.3μl Nuc(R):5'-CGTATTGCCCTTTCGAAACATT-3'(SQE ID NO:2) 0.3μl <![CDATA[H2O]]> 3.4μl

[0192] The cycle system was as follows: 95℃ for 15 min; 95℃ for 15 s; 60℃ for 20 s for 30 cycles; 72℃ for 45 s.

[0193] Figure 16As shown in Figure B, in the lysis experiment of Staphylococcus aureus ATCC29213 with endolysin protein (concentration of 400 μg / ml), 80αendolysin could reduce the absorbance at 600 nm from 1.0 to 0.2 within 60 minutes at 400 μg / ml, indicating that endolysin protein has good lysis activity.

[0194] Figure 16 C and Figure 16 As shown in Figure D, when the Ct Threshold in qPCR is set to 2000, the Ct values ​​for the endolysin + water boiling method and the water boiling method are 10 and 15, respectively. This indicates that the extraction yield of Staphylococcus aureus genomic DNA by the endolysin + water boiling method is much higher than that by the water boiling method. This is because the cell wall of Staphylococcus aureus is thick, and the ordinary water boiling method cannot effectively break down the cell wall of Staphylococcus aureus, resulting in low genomic DNA extraction efficiency. Endolysin is a peptidoglycan hydrolase encoded by bacteriophages, which can specifically lyse the host bacterial cell wall, thereby releasing intracellular substances. Therefore, the extraction yield is higher when endolysin is used in combination with the water boiling method than when the water boiling method is used alone, which plays a very important role in subsequent rapid detection.

[0195] Example 9: Amplification and verification of a specific genomic nuc fragment from Staphylococcus aureus.

[0196] The experimental method for amplifying the Staphylococcus aureus genome nuc fragment using RPA technology is as follows.

[0197] (I) Preparation of EGFP-amidase 3-CBD-Ni magnetic beads

[0198] 1. Take 100 μl of the mixed Ni-polymethyl methacrylate magnetic beads into an EP tube, wash twice with 500 μl of T100 for 2 min each time, discard the supernatant, and suspend in 100 μl of T100.

[0199] 2. Add 100 μl of EGFP-amidase 3-CBD protein at a concentration of 1 mg / ml and incubate for 20 min. Every 2 min, tilt the EP tube 50° and gently rotate it by hand.

[0200] 3. Place the incubated EGFP-amidase 3-CBD protein suspension on a magnetic rack and let it stand to remove the supernatant. Wash twice with 500 μl of 50 mM Tris-HCl, and finally resuspend in 100 μl of 50 mM Tris-HCl.

[0201] (II) Capture of Staphylococcus aureus using EGFP-amidase 3-CBD-Ni magnetic beads.

[0202] 4. Centrifuge 3 ml of Staphylococcus aureus, wash once with 1 ml of Tris-HCl, and then adjust to OD value with Tris-HCl. 600 =0.6, then diluted to 1×10 4 CFU / ml, 1×10 3 CFU / ml, 1×10 2 CFU / ml, 1×10 1 CFU / ml was used to obtain four dilutions of bacterial culture.

[0203] 5. Take 1 ml of each of the four dilutions of the above bacterial culture into EGFP-amidase 3-CBD protein suspension and incubate for 20 min. Every 2 min, tilt the EP tube at 50° and gently rotate it by hand.

[0204] 6. Place the incubated EGFP-amidase 3-CBD protein-Staphylococcus aureus suspension on a magnetic rack, discard the supernatant, add 500 μl of 50 mM Tris-HCl and wash twice, 2 min each time, then resuspend in 40 μl of 50 mM Tris-HCl.

[0205] (III) Extraction of Staphylococcus aureus DNA

[0206] 7. Add 10 μl of 2 mg / ml endolysin protein (see step 1 of Example 8 for details), incubate for 30 min, then place in a boiling water bath for 10 min, and aspirate the supernatant to obtain the crude extract.

[0207] (iv) Amplification of the NUC fragment of the Staphylococcus aureus genome

[0208] 8. Perform RPA on the crude extract. The specific steps are as follows:

[0209] The primers used in the RPA reaction were designed using Primer Premier 5.0, with a primer length between 30 and 35 nt.

[0210] Among them, RPA-1-F: 5'-GCATCACAAACAGATAACGGCGTAAATAGAAG-3' (SQE ID NO: 3);

[0211] RPA-1-R: 5'-ACATTAATTTAACCGTATCACCATCAATCGCT-3' (SQE ID NO: 4);

[0212] RPA-2-F: 5'-CATCACAAACAGGTAACGGCGTAAATAGAAGT-3' (SQE ID NO: 5);

[0213] RPA-2-R: 5'-TCTCTACACCTTTTTTAGGATGCTTTGTTTCA-3' (SQE ID NO: 6).

[0214] The RPA reaction system consisted of: 10 μM upstream primer (RPA-1-F or RPA-2-F) and 10 μM downstream primer (RPA-1-R or RPA-2-R), 50 mM Tris-HCl (pH 7.5), 100 mM potassium acetate, 14 mM magnesium acetate, 2 mM dithiothreitol (DTT), 5% polyethylene glycol, 200 μM dNTPs, 3 mM ATP, 1 μl 5000 units / ml Bsu DNA polymerase, and 2 μl of amplification product. The mixture was incubated in a standard water bath at 37°C for the RPA reaction. All concentrations in the mixture were final concentrations. RPA-1-F and RPA-1-R amplified nuc fragment 1, and RPA-2-F and RPA-2-R amplified nuc fragment 2.

[0215] The experimental method for RPA amplification of Escherichia coli genome nuc fragments is the same as that for RPA amplification of Staphylococcus aureus genome nuc fragments, the only difference being the choice of bacterial culture.

[0216] The amplification products of Staphylococcus aureus and Escherichia coli were extracted with phenol / chloroform, respectively, and subjected to 1% agarose gel electrophoresis. The results are shown below. Figure 17 .

[0217] Figure 17 The results showed that clear bands were visible in the Staphylococcus aureus lane, while no bands were visible in the Escherichia coli lane. This indicates that the nuc gene is a Staphylococcus aureus-specific gene and can be amplified by RPA isothermal amplification.

[0218] like Figure 18 As shown, for RPA sensitivity, isothermal amplification of RPA was performed using 10-fold serially diluted Staphylococcus aureus genomic DNA as a template. Agarose gel electrophoresis showed that the detection limit of RPA was 10. 2 aM.

[0219] The above experimental results show that this embodiment successfully amplified the Staphylococcus aureus-specific nuc gene fragment.

[0220] Example 10: Detection of Staphylococcus aureus using EGFP-RBP-Ni-NTA magnetic beads or EGFP-amidase 3-CBD-Ni magnetic beads

[0221] 1. Specificity assessment of the EGFP-amidase 3-CBD-Ni magnetic bead-binding recombinase polymerase amplification (RPA) method for detecting Staphylococcus aureus.

[0222] The EGFP-amidase 3-CBD-Ni magnetic beads obtained in Example 2 were used to capture and extract Staphylococcus aureus genomic DNA from the sample. 1×10 4 Staphylococcus aureus contaminated with Tris-HCl, milk, orange juice, and cheese at CFU / ml was used to bind and capture Staphylococcus aureus using EGFP-amidase 3-CBD-Ni magnetic beads obtained in Example 2. Genomic DNA was extracted using endolysin and boiling in water, followed by isothermal amplification of the nuc fragment using RPA (see Example 9 for details), and gel electrophoresis on a 1% agarose gel. Figure 19 As shown.

[0223] The results showed that contaminated Tris-HCl, milk, orange juice, and cheese could all successfully amplify the nuc gene fragment, and the bands were almost identical in brightness.

[0224] The above experimental results show that the magnetic beads of the present invention are not affected by the food matrix when capturing Staphylococcus aureus.

[0225] 2. Specificity assessment of the EGFP-RBP-Ni-NTA magnetic bead-binding PCR method for detecting Staphylococcus aureus

[0226] 2.1 Take 1 mL of 1×10 7 CFU / mL Staphylococcus aureus and Escherichia coli, added to 1.2 × 10⁻⁶ CFU / mL of the strain obtained in Example 5. 5 EGFP-RBP-Ni magnetic beads were enriched and separated (see step 3 of Example 7 for details), and finally resuspended in 50 μL of 50 mM Tris-HCl. Genomic DNA was then extracted using the boiling method (boiling water treatment for 10 min, see step 1 of Example 8 for details). Using the extracted genomic DNA as a template, the Staphylococcus aureus-specific gene nuc was amplified by PCR to verify the specificity of EGFP-RBP-Ni magnetic beads in capturing Staphylococcus aureus.

[0227] The PCR primers were Nuc R: CGTATGCCCTTTCGAAACATT (SEQ ID NO:2) and Nuc F: GGCATATGTATGGCAATTGTTTC (SEQ ID NO:1). The PCR system is shown in the table below:

[0228]

[0229] 1 μL each of forward and reverse primers were used. The mixture was pre-denatured at 98 °C for 5 min, denatured at 98 °C for 10 s, annealed at 60 °C for 5 s, and extended at 72 °C for 10 s for 30 cycles. The final extension was performed at 72 °C for 5 min, and the process was terminated at 4 °C.

[0230] like Figure 20 As shown in A, lane 1 is the product of the nuc gene fragment amplified under PCR reaction conditions after Staphylococcus aureus was captured by EGFP-RBP-Ni-NTA magnetic beads.

[0231] The results showed that EGFP-RBP-Ni magnetic beads can effectively enrich and isolate Staphylococcus aureus, and can be further used for PCR detection of Staphylococcus aureus in test samples.

[0232] 2.2 Sensitivity evaluation of the EGFP-RBP-Ni magnetic bead-binding PCR method for detecting Staphylococcus aureus

[0233] Take the 1.2×10 obtained in Example 5 5 EGFP-RBP-Ni-NTA magnetic beads were added to 1 mL of different concentrations (0, 1×10⁻⁶). 3 1×10 4 1×10 5 1×10 6 1×10 7 Staphylococcus aureus (CFU / mL) was enriched and isolated (see step 3 of Example 7 for details), and resuspended in 50 μL of 50 mM Tris-HCl. Genomic DNA was then extracted using the boiling method (boiling water treatment for 10 min, see step 1 of Example 8 for details). Using the extracted genomic DNA as a template, the Staphylococcus aureus-specific nuc gene fragment was amplified by PCR to examine the sensitivity of the EGFP-RBP-Ni magnetic bead method for detecting Staphylococcus aureus.

[0234] The results are as follows Figure 20 B shows that when the amount of EGFP-RBP-Ni-NTA magnetic beads is 1.2 × 10⁻⁶, 5 When the sample volume is 1 mL, the minimum detectable concentration of Staphylococcus aureus in the sample is 10. 3CFU / mL. The above experimental results show that the method of the present invention has good sensitivity.

[0235] 2.3 Research on the application of magnetic beads in capturing Staphylococcus aureus in the quality detection of actual food samples

[0236] The main challenge at present is whether it can be universally applied in different food environments. Therefore, the inventors contaminated Staphylococcus aureus with Tris-HCl, milk, and orange juice to a concentration of 1×10⁻⁶. 3 Using EGFP-RBP-Ni-NTA magnetic beads prepared in Example 5, Staphylococcus aureus was captured in the contaminated food sample at CFU / mL. Genomic DNA was then extracted by boiling in water, and the Staphylococcus aureus-specific nuc gene fragment was amplified by PCR. The method in step 2.2 of this example was followed, and the sample was subjected to 1% agarose gel electrophoresis.

[0237] Experimental results are as follows Figure 21 As shown, the nuc gene fragment was amplified in Tris-HCl, milk, and orange juice contaminated with Staphylococcus aureus, and the band brightness of the samples was basically the same. This indicates that the capture of Staphylococcus aureus by EGFP-RBP-Ni-NTA magnetic beads is not affected by the food matrix and can be widely used for the quality detection of Staphylococcus aureus in food, with a detection limit of 1×10⁻⁶. 3 CFU / mL.

[0238] Example 11: Detection of Staphylococcus aureus in milk by magnetic bead capture combined with cas12a / crRNA cleavage

[0239] 1. Three different crRNAs were selected (see...) Figure 23 A) The nuc fragment 1 and nuc fragment 2 obtained in Example 9 were subjected to cas12a / crRNA cleavage detection. The cas12a / crRNA cleavage detection method is as follows:

[0240] The amplification product obtained in Example 7 was mixed with 500 nM crRNA, 250 nM Cas12a, 2.5 μM ssDNA-FQ reporter (sequence 5'-FAM-TTATT-BHQ-1-3'), 2 μL NEB buffer 3.1, and 3 μL, and ddH2O was added to a final volume of 20 μL. The reaction was performed at 37°C for 30 min using a qPCR machine, with fluorescence measurements taken every 1 min. For detailed results, please refer to [link to relevant documentation]. Figure 22-23 Among them, crRNA-1 is used to recognize nuc fragment 1, and crRNA-2 and crRNA-3 are used to recognize nuc fragment 2. The crRNAs are shown below:

[0241]

[0242]

[0243] like Figure 23 As shown in Figure C, the strongest fluorescence value was produced when crRNA-1 activated cas12a / crRNA cleaved ssDNA-FQ reporter, indicating that crRNA-1 is the optimal choice among the three crRNAs. Figure 23 As shown in B, the analysis of different components in the cas12a / crRNA cleavage reaction shows that fluorescence can only be generated when all solvents in the reaction system are added; none of them can be omitted.

[0244] 2. In order to quickly detect Staphylococcus aureus in food and prevent foodborne illnesses caused by Staphylococcus aureus, the upstream EGFP-amidase 3-CBD-Ni magnetic beads obtained in Example 2 were used to capture Staphylococcus aureus, and the downstream RPA and cas12a cleavage detection were combined. Under ultraviolet light irradiation, the contamination of food was visually observed and evaluated, and the specificity and sensitivity of the method were investigated.

[0245] Experimental Method: 500 nM crRNA-1 (see step 1 of this example for details), 250 nM Cas12a, 2.5 μM ssDNA-FQ reporter (sequence 5'-FAM-TTATT-BHQ-1-3'), 2 μL NEB buffer 3.1, and 3 μL of the RPA amplification product obtained in Example 9 (containing the Staphylococcus aureus genome nuc fragment) were mixed, and ddH2O was added to a final volume of 20 μL. The reaction was carried out at 37°C for 30 min using a qPCR machine, with fluorescence measurements performed every 1 min.

[0246] Specificity study and results: 1×10 4 CFU / ml of sterile milk contaminated with Escherichia coli and Staphylococcus aureus was used to capture Staphylococcus aureus in the sample, extract Staphylococcus aureus genomic DNA, and amplify the Staphylococcus aureus genomic nuc fragment using RPA. The specificity of Staphylococcus aureus was detected by cas12a / crRNA cutting detection.

[0247] like Figure 24 As shown, Staphylococcus aureus activates cas12a and cleaves ssDNA-FQ reporter to produce fluorescence.

[0248] The above experimental results show that the EGFP-amidase 3-CBD-Ni magnetic beads of the present invention can capture Staphylococcus aureus and, combined with the cas12a / crRNA cleavage detection method, can specifically detect Staphylococcus aureus in food.

[0249] Sensitivity assessment and results: 1×10 3 CFU / ml, 1×10 2 CFU / ml, 1×10 1 CFU / ml of sterile milk contaminated with Staphylococcus aureus was collected, and then 100 μl of EGFP-amidase 3-CBD-Ni magnetic beads were added. Staphylococcus aureus genomic DNA was extracted using the endolysin + water boiling method. After RPA reaction at 37°C for 30 min (see Example 9 for details), Cas12a was digested at 37°C for 30 min. Fluorescence signals were visually examined under UV light. Data were analyzed using GraphPadPrism 8.0 software and presented as mean ± standard deviation. Differences between data means were analyzed using a t-test, with P < 0.05 indicating statistical significance.

[0250] like Figure 25 As shown in Figures A, B, and C, these represent the methods for detecting 1×10⁻⁶ ions in milk using EGFP-amidase 3-CBD-Ni magnetic bead capture combined with cas12a / crRNA cleavage. 3 CFU / ml, 1×10 2 CFU / ml, 1×10 1 CFU / ml Staphylococcus aureus. The curve in the figure is the fluorescence accumulation curve of the cutting reaction in a qPCR instrument set at 37℃ for 30 min. The test tube image in the curve is the corresponding fluorescence image under ultraviolet light after cutting at 37℃ for 30 min.

[0251] The results showed that the detection limit of EGFP-amidase 3-CBD-Ni magnetic bead capture combined with cas12a / crRNA cleavage detection could be further reduced to 1×10⁻⁶. 1 CFU / ml.

[0252] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0253] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for detecting Staphylococcus aureus in food samples, characterized in that, include: The sample to be tested was treated by contact with adsorption magnetic beads, which included Ni-polymethyl methacrylate magnetic beads and binding protein. The N-terminus of the binding protein was connected to the Ni-polymethyl methacrylate magnetic beads. The binding protein was attached with a fluorescent group. The binding protein was Amidase 3-CBD protein derived from Staphylococcus aureus phage 80α. The amino acid sequence of the Amidase 3-CBD protein is shown in SEQ ID NO:

14. The contact treatment product was subjected to DNA extraction. The endolysin and the contact treatment product were reacted for 20-40 minutes. The contact treatment product and the endolysin were then boiled in water for 5-20 minutes. The volume ratio of the endolysin to the sample to be tested was 1:(40-60), and the concentration of the endolysin was 1-3 mg / ml. The endolysin was selected from the phage endolysin derived from the Staphylococcus aureus. The DNA extraction product was subjected to recombinase polymerase amplification. The nucleotide sequences of the primers used for recombinase polymerase amplification are shown in SEQ ID NO:3 and 4. The amplification product, Cas12a, crRNA, and ssDNA-FQ reporter were subjected to a third mixing process, and the nucleotide sequence of the crRNA is shown in SEQ ID NO:7; Based on the fluorescence signal generated by the third mixing process, the presence of Staphylococcus aureus or the content of Staphylococcus aureus in the sample to be tested is detected.

2. The method according to claim 1, characterized in that, The fluorescent group is the EGFP group.

3. The method according to claim 1, characterized in that, The adsorption magnetic beads further include protein tags.

4. The method according to claim 3, characterized in that, The C-terminus of the protein tag is connected to the N-terminus of the binding protein, and the N-terminus of the protein tag is connected to the Ni-polymethyl methacrylate magnetic bead.

5. The method according to claim 3, characterized in that, The protein tag is selected from at least one of the following: His tag, Flag tag, GST tag, MBP tag, SUMO tag, and C-Myc tag.

6. The method according to claim 3, characterized in that, The protein tag is a His tag.

7. The method according to claim 1, characterized in that, The adsorption magnetic beads are obtained by first mixing Ni-polymethyl methacrylate magnetic beads and binding protein.

8. The method according to claim 7, characterized in that, The first mixing treatment was carried out at a temperature of 2-30°C and a pH of 5-9.

9. The method according to claim 1, characterized in that, Prior to the contact treatment, NaCl and the adsorbent magnetic beads are subjected to a second mixing treatment.

10. The method according to claim 9, characterized in that, The final concentration of NaCl is 50~200mM.

11. The method according to claim 1, characterized in that, The number of adsorption magnetic beads added is not less than 1.0 × 10⁻⁶. 5 indivual.

12. The method according to claim 1, characterized in that, The third mixing process generates a fluorescent signal, which is an indication that the sample to be tested contains Staphylococcus aureus; or The third mixing treatment does not produce a fluorescence signal, which is an indication that the sample to be tested does not contain Staphylococcus aureus.

13. The method according to claim 1, characterized in that, The detection further includes: The content of Staphylococcus aureus in the sample to be tested is determined based on the standard curve, which is the curve corresponding to a predetermined amount of Staphylococcus aureus and the intensity of the fluorescence signal.

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