CN116287139A - Method for detecting staphylococcus aureus - Google Patents
Method for detecting staphylococcus aureus Download PDFInfo
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- CN116287139A CN116287139A CN202211716362.1A CN202211716362A CN116287139A CN 116287139 A CN116287139 A CN 116287139A CN 202211716362 A CN202211716362 A CN 202211716362A CN 116287139 A CN116287139 A CN 116287139A
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- staphylococcus aureus
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Abstract
The invention provides an adsorption magnetic bead. The adsorption magnetic bead comprises: ni-polymethacrylate magnetic beads; and the binding protein is used for binding staphylococcus aureus, and the Ni-polymethacrylate magnetic beads are connected with the binding protein. In the invention, the binding protein in the adsorption magnetic beads can be combined with staphylococcus aureus, the adsorption magnetic beads are added into a sample to be detected, and the staphylococcus aureus is combined through the binding protein, so that the capture and enrichment of the adsorption magnetic beads on the staphylococcus aureus can be realized, and the subsequent detection of the staphylococcus aureus is facilitated. The method for detecting the adsorption magnetic beads, the kit and the staphylococcus aureus has the advantages of reduced strain culture steps, short detection time and practically improved detection efficiency.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a method for detecting staphylococcus aureus, and more particularly relates to an adsorption magnetic bead and application, a kit and application thereof, and a method for detecting staphylococcus aureus.
Background
Staphylococcus aureus (s. Aureus) is widely distributed in nature, and is one of the main pathogens causing human and animal infections, which easily contaminates foods such as milk, vegetables, and the like. Staphylococcus aureus can produce virulence factors including enterotoxins, desquamation toxins, toxic shock syndrome toxin-1 and ubiquitin-valien Ding Bai cytokines associated with toxic shock syndrome, food poisoning and severe allergic diseases. Staphylococcus aureus has been reported to be widely present in raw milk and dairy products. In recent years, staphylococcus aureus has become the third largest food-borne microbial pathogen after salmonella and vibrio parahaemolyticus. Staphylococcus aureus-induced food poisoning events occur due to the lack of an effective, rapid and visual detection method.
Therefore, it is highly desirable to establish a rapid, sensitive, mass-applicable detection method.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems existing in the prior art to at least some extent. Therefore, the invention provides a method for detecting staphylococcus aureus, which can effectively detect staphylococcus aureus.
The present invention has been completed based on the following findings by the inventors:
several methods for rapid detection and identification of staphylococcus aureus in food samples, such as Polymerase Chain Reaction (PCR), enzyme-linked immunosensor, and nucleic acid-based molecular biology methods, have been developed today, these advanced microbial detection methods shorten the detection time from days to hours, but still suffer from limitations, e.g., these methods are generally expensive, require high-tech laboratory equipment and skilled technicians, and cannot be used as in-situ detection methods. Furthermore, in remote areas, the lack of experimental equipment severely limits the detection of food borne pathogens.
Phages are viruses of a specific infectious bacterium, also known as "bacterial viruses", which are generally between 20 and 200nm in size, short in life cycle (about 20min to 60 min), and are recognized as the most abundant biological entities on earth, up to about 10 31 There may also be bacterial phages present in the bacteria, which play an important role in the ecological balance of the microorganism's life. The primary structure of phage can be divided into a nucleocapsid or head, a complex tail structure and a base. The head capsid and the tail are composed of proteins, and the head capsid contains the genetic material of phage, nucleic acid DNA or RNA. The capsid attaches to the tail with fibers for recognition and adhesion to receptors on the bacterial cell surface.
The immune magnetic bead separation technology (IMS) is a technology combining an immunological technology with magnetic beads, and the principle is that a specific antibody is combined with the magnetic beads to prepare the immune magnetic beads, and then the immune magnetic beads are combined with a specific antigen in a mixed solution, so that a target antigen and the rest impurities are thoroughly separated through magnetic separation, and the aim of enrichment and separation is fulfilled. Compared with the traditional method, the method is quick and simple, and can improve the sensitivity and the authenticity of detection.
Based on this, in one aspect of the present invention, the present invention proposes an adsorption magnetic bead. According to an embodiment of the present invention, the adsorption magnetic beads include: ni-polymethacrylate magnetic beads; and the binding protein is used for binding staphylococcus aureus, and the Ni-polymethacrylate magnetic beads are connected with the binding protein.
The inventors have also found that the binding proteins in the adsorbent magnetic beads bind (in particular bind specifically) to staphylococcus aureus. The adsorption magnetic beads are added into a sample to be detected, staphylococcus aureus can be combined through the binding protein, the capture and enrichment of the magnetic beads to the staphylococcus aureus can be further realized through the magnetic separation effect, the subsequent detection of the staphylococcus aureus can be facilitated, the strain culture steps are reduced, the detection time is shortened, and the detection efficiency is improved.
In another aspect of the invention, the invention provides the use of the adsorption magnetic beads in the preparation of a kit for detecting staphylococcus aureus. From the previous results, the binding proteins in the adsorption magnetic beads can bind staphylococcus aureus, so that the adsorption magnetic beads can capture and enrich staphylococcus aureus, and the staphylococcus aureus can be separated and obtained. Therefore, the kit containing the adsorption magnetic beads can detect staphylococcus aureus, and has the advantages of short detection time, high detection efficiency and the like.
In yet another aspect of the invention, the invention provides a kit. According to an embodiment of the invention, the kit comprises: the aforementioned adsorption magnetic beads. From the previous results, the binding proteins in the adsorption magnetic beads can bind staphylococcus aureus, so that the adsorption magnetic beads can capture and enrich staphylococcus aureus, and the staphylococcus aureus can be separated and obtained. Therefore, the kit containing the adsorption magnetic beads can detect staphylococcus aureus, and has the advantages of short detection time, high detection efficiency and the like.
In a further aspect of the invention, the invention proposes the use of the aforementioned adsorption magnetic beads or the aforementioned kit for the preparation of a product for the detection of staphylococcus aureus. The prior art shows that the binding protein in the adsorption magnetic beads can bind staphylococcus aureus, so that the adsorption magnetic beads can capture and enrich the staphylococcus aureus, and the staphylococcus aureus can be separated and obtained; and the kit containing the adsorption magnetic beads can capture and enrich staphylococcus aureus so as to separate and obtain staphylococcus aureus. Therefore, the product of the invention can detect staphylococcus aureus, and has the advantages of short detection time, high detection efficiency and the like.
In yet another aspect of the invention, the invention provides a method of detecting staphylococcus aureus. According to an embodiment of the invention, the method comprises: the adsorption magnetic beads or the kit are adopted to carry out contact treatment on a sample to be detected so as to detect whether staphylococcus aureus or the content of the staphylococcus aureus is contained in the sample to be detected. As described above, the adsorption magnetic beads or the kit can be used for combining staphylococcus aureus to capture and enrich the staphylococcus aureus, so that the method can detect the staphylococcus aureus without culturing the strain, and has the advantages of simplified detection steps, short detection time, high detection efficiency and the like.
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.
Drawings
The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:
FIG. 1 is a schematic diagram of the different domains and cloning fragments of 80. Alpha. Endolysin according to example 1 of the present invention;
FIG. 2 is a diagram showing the purification of 80. Alpha. Endolysin different domain proteins according to example 2 of the present invention;
FIG. 3 is a graph showing the results of the fluorescence microscope and flow cytometer detection for binding of different proteins to Staphylococcus aureus in example 3 of the present invention;
FIG. 4 is a graph showing the results of Western detection on binding of different proteins to Staphylococcus aureus in example 3 of the present invention;
FIG. 5 shows the results of the stability analysis of EGFP-amidase 3-CBD protein at different concentrations of NaCl in example 4 of the present invention;
FIG. 6 shows the results of the analysis of EGFP-amidase 3-CBD protein stability at different temperatures in example 4 of the present invention;
FIG. 7 shows the results of the stability of EGFP-amidase 3-CBD protein at different pH values in example 4 of the present invention;
FIG. 8 shows a double-restriction map of the recombinant plasmid pET28a-EGFP-RBP of example 5 of the present invention, wherein (A) is a double-restriction map of PMV-RBP and pET28a-EGFP, and (B) is a double-restriction map of pET28 a-EGFP-RBP;
FIG. 9 shows SDS-PAGE patterns after EGFP-RBP protein purification in example 5 of the present invention, wherein (A) is an EGFP protein electrophoresis pattern and (B) is an EGFP-RBP protein electrophoresis pattern;
FIG. 10 shows the binding of EGFP-RBP to Staphylococcus aureus in example 6 of the present invention, wherein (A) is the fluorescence microscopic imaging result, (B) is the quantitative analysis result of the flow cytometer, and (C) is the Western Blot result;
FIG. 11 shows the result of fluorescence microscopy of EGFP-RBP-Ni magnetic bead preparation in example 7 of the present invention;
FIG. 12 is a graph showing the result of capturing Staphylococcus aureus by EGFP-amidase3-CBD-Ni magnetic beads in example 7 of the present invention, wherein (A) is the result of observing EGFP-amidase3-CBD-Ni magnetic beads under a fluorescence microscope, and (B) is the result of observing the bright-dark field of EGFP-amidase3-CBD-Ni magnetic beads under a fluorescence microscope;
FIG. 13 is a schematic diagram showing EGFP-amidase3-CBD-Ni magnetic bead capture and recovery efficiency in example 7 of the present invention;
FIG. 14 is a sensitivity analysis of EGFP-RBP-Ni-NTA magnetic beads of example 7 of the present invention;
FIG. 15 is a graph showing the capturing efficiency of EGFP-RBP-Ni-NTA magnetic beads in example 7 of the present invention, wherein (A) is a graph showing the capturing efficiency of different EGFP-RBP protein concentrations, and (B) is a graph showing the capturing efficiency of different numbers of Ni magnetic beads;
FIG. 16 shows the results of an analysis of the genomic DNA of Staphylococcus aureus extracted by endolysin + water-boiling method and water-boiling method according to example 8 of the present invention;
FIG. 17 is an agarose gel electrophoresis of different bacteria after nuc fragments have been amplified by the RPA method of example 9 of the present invention;
FIG. 18 is an agarose gel electrophoresis of the RPA method of example 9 of the invention after amplification of genomic DNA from Staphylococcus aureus at different concentrations;
FIG. 19 is an agarose gel electrophoresis of samples from different sources of Staphylococcus aureus genomic DNA amplified by the RPA method of example 10 of the present invention;
FIG. 20 shows EGFP-RBP-Ni magnetic bead binding PCR specificity assay (A) and EGFP-RBP-Ni magnetic bead binding PCR sensitivity assay (B) of example 10 of the present invention;
FIG. 21 shows EGFP-RBP-Ni magnetic bead binding PCR detection of Staphylococcus aureus in different matrices according to example 10 of the present invention, wherein lane 1 is Tris-HCl, lane 2 is orange juice, and lane 3 is milk;
FIG. 22 shows agarose gel electrophoresis of crRNA-1, crRNA-2, and crRNA-3 purified in example 11 of the present invention;
FIG. 23 is a schematic representation of cas12a/crRNA cleavage assay and analysis of the assay results in example 11 of the present invention;
FIG. 24 is a specific assay for EGFP-amidase 3-CBD-Ni magnetic bead capture binding cas12a/crRNA cleavage assay in example 11 of the present invention;
FIG. 25 shows the result of EGFP-amidase 3-CBD-Ni magnetic bead capture binding cas12a/crRNA cleavage assay in example 11 of the present invention.
Detailed Description
Embodiments of the present invention are described in detail below. The following examples are illustrative only and are not to be construed as limiting the invention.
It should be noted that the terms "first," "second," and "second" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implying a number of technical features being indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature. Further, in the description of the present invention, unless otherwise indicated, the meaning of "a plurality" is two or more.
The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.
In this document, the terms "comprise" or "include" are used in an open-ended fashion, i.e., to include what is indicated by the present invention, but not to exclude other aspects.
In this document, the terms "optionally," "optional," or "optionally" generally refer to the subsequently described event or condition may, but need not, occur, and the description includes instances in which the event or condition occurs, as well as instances in which the event or condition does not.
The invention provides an adsorption magnetic bead, application thereof, a kit and application thereof and a method for detecting staphylococcus aureus, and the adsorption magnetic bead, the kit and the application thereof and the method for detecting staphylococcus aureus are respectively described in detail below.
Adsorption magnetic bead
In one aspect of the invention, the invention provides an adsorptive magnetic bead. According to an embodiment of the present invention, the adsorption magnetic beads include: ni-polymethacrylate magnetic beads; and the binding protein is used for binding staphylococcus aureus, and the Ni-polymethacrylate magnetic beads are connected with the binding protein.
The inventor also finds that the binding protein in the adsorption magnetic beads can bind (especially can specifically bind) staphylococcus aureus, and the adsorption magnetic beads are added into a sample to be detected, so that the adsorption magnetic beads can bind the staphylococcus aureus, capture and enrichment of the staphylococcus aureus can be realized, the subsequent detection of the staphylococcus aureus is facilitated, the strain culture steps are reduced, the detection time is shortened, and the detection efficiency is improved.
According to an embodiment of the present invention, the above-mentioned adsorption magnetic bead may further include at least one of the following technical features:
according to an embodiment of the invention, the N-terminus of the binding protein is linked to the Ni-polymethacrylate magnetic beads.
As used herein, the term "attached" may be either directly to the binding protein or indirectly to the Ni-polymethacrylate beads, and is not particularly limited. The binding protein is attached to the Ni-polymethacrylate beads, for example, by a protein tag.
According to an embodiment of the invention, the magnetic adsorption beads further comprise a protein tag.
In some alternative embodiments of the invention, the Ni-polymethacrylate magnetic beads are linked to the binding protein via a His-tag sequence.
According to an embodiment of the invention, the C-terminal of the protein tag is connected with the N-terminal of the binding protein, and the N-terminal of the protein tag is connected with the Ni-polymethacrylate magnetic beads.
According to an embodiment of the invention, the protein tag comprises at least one selected from the group consisting of His tag, flag tag, GST tag, MBP tag, SUMO tag and C-Myc tag.
According to an embodiment of the invention, the protein tag is a His tag.
According to an embodiment of the invention, the Ni-polymethacrylate magnetic beads and the binding proteins are provided in a manner of combining Ni and His-tag sequences.
According to an embodiment of the invention, the binding protein is derived from a staphylococcus aureus bacteriophage. The inventor further discovers that the binding protein from staphylococcus aureus phage, especially the receptor binding protein and/or Amidase protein from staphylococcus aureus phage can effectively realize the capture and enrichment of Ni-polymethacrylate magnetic beads on staphylococcus aureus, further shortens the operation time of the subsequent bacteria detection step, and further improves the detection efficiency.
In a specific embodiment, the binding protein is an endolysin of a staphylococcus aureus phage or a truncate comprising the cell wall binding domain (C-terminal cell wall binding domain, CBD) of said endolysin.
In a specific embodiment, the binding protein may be Staphylococcus aureus phage endolysin SA97 (LysSA 97) or a truncation of a LysSA 97-containing cell wall binding domain protein. LysSA97 specifically cleaves staphylococcus aureus strains and causes their biofilm to be disrupted, having a specific cell wall binding domain (CBD) and two Enzyme Activity Domains (EAD), containing cysteine, histidine-dependent amidohydrolase/peptidase (CHAP, PF 05257) and N-acetylmuramyl-L-alanine-nine amidase (amidase-3, pf01520) domains.
In a specific embodiment, the binding protein may be Staphylococcus aureus phage 80 alpha or a truncated form comprising its central Amidase 3 catalytic domain (also known as Amidase 3, amidase_3 or Amidase-3) and a C-terminal SH3b cell-binding domain (abbreviated as SH3 b).
According to an embodiment of the invention, the binding protein may also be a receptor binding protein of Staphylococcus aureus phage (Receptor binding protein, RBP for short).
According to an embodiment of the invention, the binding protein is derived from staphylococcus aureus phage 80 a. The inventor finds that the protein derived from the staphylococcus aureus phage 80 alpha has strong binding capacity with staphylococcus aureus, so that the protein and the Ni-polymethacrylate magnetic beads are used for preparing Cheng Xifu magnetic beads, staphylococcus aureus in a sample to be detected can be effectively captured, and the accuracy of detecting staphylococcus aureus in the sample to be detected can be improved.
In some specific embodiments, the Amidase protein of the Staphylococcus aureus phage is an Amidase 3-CBD protein. In a more specific embodiment, the staphylococcus aureus phage is staphylococcus aureus phage 80 a.
Amidase protein or Amidase 3-CBD protein in the present invention specifically refers to Amidase enzyme derived from Staphylococcus aureus phage. Amidase enzyme, also known as n-acetylmuramyl-L-alanine Amidase (NAMLAAEC), is a PGN hydrolase capable of specifically cleaving an amide bond between the lactoyl group of muramic acid and the alpha-amino group of L-alanine. In phages, the Amidase enzymes have a critical role in the lytic cycle of the virus, they are responsible for host cell lysis, allowing release and transmission of phage progeny. The invention utilizes the combination property of staphylococcus aureus phage Amidase protein and staphylococcus aureus, skillfully realizes the specific capture and enrichment of magnetic beads on staphylococcus aureus through the Amidase protein, and further provides possibility for further improving and enhancing the existing method for detecting and identifying staphylococcus aureus in food samples.
According to an embodiment of the invention, the binding protein comprises at least one selected from the group consisting of a receptor binding protein derived from Staphylococcus aureus phage 80 alpha and an Amidase 3CBD protein.
The inventors have unexpectedly found that the receptor binding protein derived from staphylococcus aureus phage 80 alpha and the Amidase 3CBD protein have stronger binding ability to staphylococcus aureus than other binding proteins, and therefore, the inventors prepared RBP and/or Amidase 3CBD proteins with Ni-polymethacrylate magnetic beads to obtain adsorption magnetic beads, and used the adsorption magnetic beads for detection of staphylococcus aureus, and have the advantages of strong stability, ligand specificity, strong affinity to carbohydrate epitopes, high sensitivity, and the like, compared with other technologies (e.g., antibodies).
According to an embodiment of the invention, the receptor binding protein has the amino acid sequence shown as SEQ ID NO. 13.
MDNKLITDLSRVFDYRYVDENEYNFKLISDMLTDFNFSLEYHRNKEVFAHNGEQIKYEHLNVTSSVSDFLTYLNGRFSNMVLGHNGDGINEVKDARVDNTGYDHKTLQDRLYHDYSTLDAFTKKVEKAVDENYKEYRATEYRFEPKEQEPEFITDLSPYTNAVMQSFWVDPRTKIIYMTQARPGNHYMLSRLKPNGQFIDRLLVKNGGHGTHNAYRYIGNELWIYSAVLDANENNKFVRFQYRTGEITYGNEMQDVMPNIFNDRYTSAIYNPIENLMIFRREYKASERQLKNSLNFVEVRSADDIDKGIDKVLYQMDIPMEYTSDTQPMQGITYDAGILYWYTGDSKPANPNYLQGFDIKTKELLFKRRIDIGGVNNNFKGDFQEAEGLDMYYDLETGRKALLIGVTIGPGNNRHHSIYSIGQRGVNQFLKNIAPQVSMTDSGGRVKPLPIQNPAYLSDITEVGHYYIYTQDTQNALDFPLPKAFRDAGWFFDVLPGHYNGALRQVLTRNSTGRNMLKFERVIDIFNKKNNGAWNFCPQNAGYWEHIPKSITKLSDLKIVGLDFYITTEESNRFTDFPKDFKGIAGWILEVKSNTPGNTTQVLRRNNFPSAHQFLVRNFGTGGVGKWSLFEGKVVE(SEQ ID NO:13)。
Further, according to embodiments of the invention, the receptor binding proteins include an amino acid sequence having at least 90% homology to the amino acid sequence set forth in SEQ ID NO. 13 or an amino acid sequence having equivalent or similar Staphylococcus aureus binding properties to the receptor binding protein set forth in SEQ ID NO. 13.
According to an embodiment of the invention, the Amidase 3CBD protein has an amino acid sequence as shown in SEQ ID NO. 14. The inventor finds that the capture effect of Amidase 3-CBD protein on staphylococcus aureus is better than that of CBD protein on staphylococcus aureus through experiments.
KIMLVAGHGYNDPGAVGNGTNERDFIRKYITPNIAKYLRHAGHEVALYGGSSQSQDMYQDTAYGVNVGNKKDYGLYWVKSQGYDIVLEIHLDAAGESASGGHVIISSQFNADTIDKSIQDVIKNNLGQIRGVTPRNDLLNVNVSAEININYRLSELGFITNKNDMDWIKKNYDLYSKLIAGAIHGKPIGGLVAGNVKTSAKNKKNPPVPAGYTLDKNNVPYKKEQGNYTVANVKGNNVRDGYSTNSRITGVLPNNTTITYDGAYCINGYRWITYIANSGQRRYIATGEVDKAGNRISSFGKFSTI(SEQ ID NO:14)。
Further, according to an embodiment 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 an equivalent or similar Staphylococcus aureus binding property to the Amidase 3CBD protein shown in SEQ ID NO. 14.
According to an embodiment of the invention, the binding protein has a fluorescent group attached. Thereby facilitating protein tracking.
According to an embodiment of the invention, the fluorescent group is an EGFP group.
According to the embodiment of the invention, the adsorption magnetic beads are prepared by adopting the following method: the Ni-polymethacrylate magnetic beads and binding proteins are subjected to a first mixing treatment so as to obtain the adsorption magnetic beads.
According to an embodiment of the present invention, the binding protein is a Receptor Binding Protein (RBP) added in an amount of 20 to 80ng, and the Ni-polymethacrylate beads are added in an amount of (1.0 to 10). Times.10 5 And each.
In a preferred embodiment of the present invention, the receptor binding protein is added in an amount of 40 to 80ng, more preferably 5 to 60ng.
In a preferred embodiment of the present invention, the Ni-polymethacrylate magnetic beads are added in an amount of (1.0 to 2.0). Times.10 5 And each.
Kit for detecting a substance in a sample
In another aspect of the invention, the invention provides a kit. According to an embodiment of the invention, the kit comprises: the aforementioned adsorption magnetic beads. From the previous results, the binding proteins in the adsorption magnetic beads can bind staphylococcus aureus, so that the adsorption magnetic beads can capture and enrich staphylococcus aureus, and the staphylococcus aureus can be separated and obtained. Therefore, the kit containing the adsorption magnetic beads can detect staphylococcus aureus, and has the advantages of short detection time, high detection efficiency, high detection sensitivity and the like.
The method for providing the Ni-polymethacrylate magnetic beads and the binding proteins with His-tag sequences in the kit is not strictly limited, and the Ni-polymethacrylate magnetic beads and the binding proteins with His-tag sequences can be provided independently and are contacted when the kit is used, so that the combination of the Ni-polymethacrylate magnetic beads and the binding proteins is realized; it can also be provided directly in the form of a conjugate, i.e. the Ni-polymethacrylate beads and the binding protein are bound in the form of Ni and His-tag sequences, i.e. in the form of the aforementioned adsorption beads. When the conjugate is used, the conjugate is directly contacted with a sample to be detected, so that the aim of capturing pathogens is fulfilled.
According to an embodiment of the invention, the kit further comprises: at least one of Cas12a, crRNA, and ssDNA-FQ reporter.
According to an embodiment of the invention, the kit further comprises Cas12a, crRNA and ssDNA-FQ reporter. Wherein, after the Cas12a protein binds with crRNA to form a complex, it binds with the amplified product, when the complex recognizes the target sequence in the amplified product, the Dnase region in Cas12a is activated, the target sequence fragment is cleaved off, then the single-stranded ssDNA is cleaved, and a detectable fluorescent signal is generated. Therefore, under the condition of normal temperature (30-40 ℃), the amplified product, cas12a, crRNA and ssDNA-FQ reporter are mixed and reacted, the DNA fragment in the amplified product can be rapidly detected, an instrument is not required, and the operation is simple.
Use of the same
In a further aspect of the invention, the invention provides the use of the aforementioned adsorption magnetic beads for the preparation of a kit for detecting staphylococcus aureus. From the previous results, the binding proteins in the adsorption magnetic beads can bind staphylococcus aureus, so that the adsorption magnetic beads can capture and enrich staphylococcus aureus, and the staphylococcus aureus can be separated and obtained. Therefore, the kit containing the adsorption magnetic beads can detect staphylococcus aureus, and has the advantages of short detection time, high detection efficiency, high detection sensitivity and the like.
In a further aspect of the invention, the invention proposes the use of the aforementioned adsorption magnetic beads or the aforementioned kit for the preparation of a product for the detection of staphylococcus aureus. The prior art shows that the binding protein in the adsorption magnetic beads can bind staphylococcus aureus, so that the adsorption magnetic beads can capture and enrich the staphylococcus aureus, and the staphylococcus aureus can be separated and obtained; and the kit containing the adsorption magnetic beads can capture and enrich staphylococcus aureus so as to separate and obtain staphylococcus aureus. Therefore, the product of the invention can detect staphylococcus aureus, and has the advantages of short detection time, high detection efficiency, high detection sensitivity and the like.
Method for detecting staphylococcus aureus
In yet another aspect of the invention, the invention provides a method of detecting staphylococcus aureus. According to an embodiment of the invention, the method comprises: the adsorption magnetic beads or the kit are adopted to carry out contact treatment on a sample to be detected so as to detect whether staphylococcus aureus or the content of the staphylococcus aureus is contained in the sample to be detected. As described above, the adsorption magnetic beads or the kit can be used for combining staphylococcus aureus to capture and enrich the staphylococcus aureus, so that the method can detect the staphylococcus aureus without culturing the strain, and has the advantages of simplified detection steps, short detection time, high detection efficiency, high detection sensitivity and the like.
The method of the present invention is a method for the purpose of non-disease diagnosis, and is used for detecting staphylococcus aureus in non-animal tissues such as food, articles, etc. or blood samples.
According to an embodiment of the invention, the method further comprises: carrying out DNA extraction treatment on the contact treatment product; amplifying the DNA extraction treatment product; detecting the amplification treatment product so as to detect whether staphylococcus aureus or the content of the staphylococcus aureus is contained in the sample to be detected.
According to an embodiment of the present invention, the adsorption magnetic beads are obtained by: the Ni-polymethacrylate magnetic beads and the binding proteins are subjected to a first mixing treatment.
According to an embodiment of the invention, the binding protein is Amidase 3-CBD protein, and the first mixing treatment is carried out at a temperature of (2-30) deg.C and a pH of 5-9. Thus, the capture efficiency of the adsorption magnetic beads to staphylococcus aureus can be improved.
According to an embodiment of the invention, the binding protein is an Amidase 3-CBD protein, and the NaCl and the adsorption beads are subjected to a second mixing treatment before the contacting treatment.
According to an embodiment of the invention, the final concentration of NaCl is (50-200) mM, e.g., (50-150) mM, (50-100) mM, preferably 100mM. Thus, the binding effect of the binding protein on the adsorption magnetic beads and staphylococcus aureus is better. In particular, the binding effect of Amidase 3-CBD protein to Staphylococcus aureus is continuously weakened with increasing NaCl concentration.
According to an embodiment of the invention, the number of the magnetic beads is not less than 1.0X10 5 E.g. not less than 1.1X10 5 Not less than 1.2X10) 5 Not less than 1.3X10) 5 Not less than 1.4X10) 5 Not less than 1.5X10) 5 Not less than 2.0X10) 5 Not less than 1.0X10) 6 Not less than 1.0X10) 7 And each. Therefore, the capturing capability of the binding protein of the adsorption magnetic beads to staphylococcus aureus can be further improved, and particularly, the capturing capability of RBP to staphylococcus aureus is improved.
According to an embodiment of the present invention, the DNA extraction process includes: and (3) carrying out water boiling treatment on the contact treatment product and the endolysin. The inventor finds out through a large number of experiments that endolysin can specifically lyse the cell wall of staphylococcus aureus, so that intracellular substances of staphylococcus aureus are released, then DNA in staphylococcus aureus can be extracted through water boiling, the extraction amount of staphylococcus aureus DNA can be greatly improved, and the method has the advantages of shortening the extraction time, being convenient to operate, being large in extraction amount and the like.
According to an embodiment of the invention, the endolysin and the contact treatment product are reacted for 20-40min before the water boiling treatment. Thus, the staphylococcus aureus cell wall can be fully cracked, and the extraction amount of staphylococcus aureus DNA can be increased.
According to the embodiment of the invention, the time of the water boiling treatment is 5-20min. Thus, the extraction effect of staphylococcus aureus DNA is better.
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 endolysin is (1-3) mg/ml. Thus, the extraction effect of staphylococcus aureus DNA is better.
It should be noted that, the sample to be tested may be pretreated before being treated with the above-mentioned magnetic beads or the above-mentioned kit, and the pretreatment includes, but is not limited to, dilution treatment and impurity removal treatment, and the specific manner is not limited.
According to an embodiment of the invention, the endolysin is selected from the group of bacteriophage endolysins derived from the staphylococcus aureus.
According to an embodiment of the invention, the endolysin is selected from the group consisting of bacteriophage 80 alpha endolysins (abbreviated endolysin or 80 alpha endolysin) derived from said staphylococcus aureus.
According to an embodiment of the invention, the phage 80. Alpha. Endolysin has the amino acid sequence shown in SEQ ID NO. 15.
MLMTKNQAEKWFDNSLGKQFNPDGWYGFQCYDYANMFFMLATGERLQGLYAYNIPFDNKAKIEKYGQIIKNYDSFLPQKLDIVVFPSKYGGGAGHVEIVESANLNTFTSFGQNWNGKGWTNGVAQPGWGPETVTRHVHYYDNPMYFIRLNFPNNLSVGNKAKGIIKQATTKKEAVIKPKKIMLVAGHGYNDPGAVGNGTNERDFIRKYITPNIAKYLRHAGHEVALYGGSSQSQDMYQDTAYGVNVGNKKDYGLYWVKSQGYDIVLEIHLDAAGESASGGHVIISSQFNADTIDKSIQDVIKNNLGQIRGVTPRNDLLNVNVSAEININYRLSELGFITNKNDMDWIKKNYDLYSKLIAGAIHGKPIGGLVAGNVKTSAKNKKNPPVPAGYTLDKNNVPYKKEQGNYTVANVKGNNVRDGYSTNSRITGVLPNNTTITYDGAYCINGYRWITYIANSGQRRYIATGEVDKAGNRISSFGKFSTI(SEQ ID NO:15)。
The endolysin was obtained by a conventional cloning expression method.
According to an embodiment of the invention, the amplification treatment is performed using a recombinase polymerase amplification method. Therefore, the method for amplifying (Recombinase Polymerase Amplification, called RPA for short) by adopting the recombinase polymerase can work at a low temperature and constant temperature, does not need thermal denaturation of a template, does not need an instrument, and has the advantages of simplicity in operation and the like. In particular, the specific steps and reagents required for the recombinase polymerase amplification method of the present invention are not strictly limited and can be obtained by conventional means in the art.
According to an embodiment of the invention, the detecting comprises: performing third mixing treatment on the amplification treatment product, the Cas12a, the crRNA and the ssDNA-FQ reporter; and detecting whether staphylococcus aureus or the content of the staphylococcus aureus is contained in the sample to be detected based on the fluorescent signal generated by the third mixing treatment. Wherein, after the Cas12a protein binds with crRNA to form a complex, it binds with the amplified product, when the complex recognizes the target sequence in the amplified product, the Dnase region in Cas12a is activated, the target sequence fragment is cleaved off, then the single-stranded ssDNA is cleaved, and a detectable fluorescent signal is generated. Therefore, under the condition of normal temperature (30-40 ℃), the amplified product, cas12a, crRNA and ssDNA-FQ reporter are mixed and reacted, the DNA fragment in the amplified product can be rapidly detected, an instrument is not required, and the operation is simple.
According to an embodiment of the present invention, 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 process does not generate a fluorescent signal, which is an indication that the sample to be tested does not contain staphylococcus aureus.
According to an embodiment of the invention, the detecting further comprises: and determining the content of staphylococcus aureus in the sample to be detected based on a standard curve, wherein the standard curve is a curve corresponding to the preset quantity of staphylococcus aureus and fluorescence signal intensity.
According to an embodiment of the invention, the crRNA has a nucleotide sequence as shown in SEQ ID NO. 7 or a nucleotide sequence having at least 90% or more homology thereto. Thus, the sensitivity of the detection of Staphylococcus aureus can be further improved, and the minimum detection limit can be 1×10 1 CFU/mL。
UAAUACGACUCACUAUAGGGUAAUUUCUACUAAGUGUAGAUGUUGAAGUUGCACUAUAUAC(SEQ IDNO:7)。
The scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The nucleotide sequences in the examples described below can be obtained according to their corresponding amino acid sequences using conventional methods or conventional software (e.g., on-line program vector (website: https:// www.vectorbuilder.cn/tool/code-optimization. Html), geneOptimizer on-line program, etc.).
Example 1: analysis of 80. Alpha. Endolysin bioinformatics of Staphylococcus aureus phage
The 80. Alpha. Endolysin gene was found by analysis from phage 80. Alpha. Genome, the 80. Alpha. Endolysin domain was analyzed by Interpro, and the molecular weight and isoelectric point of the protein were calculated using the calculation pI/Mw program ExpASY.
The sequence of the 80. Alpha. Endolysin gene was found from the phage 80. Alpha. Genome by analysis and searching of the functional domain by Interpro, and the results showed that the 80. Alpha. Endolysin gene consisted of three domains, an N-terminal CHAP catalytic domain (abbreviated as CHAP), a central Amidase 3 catalytic domain (also known as Amidase 3, amidase_3 or Amidase-3) and a C-terminal SH3b cell binding domain (abbreviated as SH3 b), as shown in FIG. 1.
Therefore, the invention selects two genes, namely Amidase 3-CBD and CBD, for cloning and expressing. Wherein, the amino acid sequence of phage 80 alpha Endolysin is shown as SEQ ID NO. 15, the amino acid sequence of Amidase 3-CBD is shown as SEQ ID NO. 14, and the amino acid sequence of CBD is shown as SEQ ID NO. 16.
YKKEQGNYTVANVKGNNVRDGYSTNSRITGVLPNNTTITYDGAYCINGYRWITYIANSGQRRYIATGEVDKAGNRIS SFGKFSTI(SEQ ID NO:16)。
Example 2: cloning, expression and purification of EGFP-Amidase 3-CBD and EGFP-CBD proteins
Cloning nucleotide sequences corresponding to Amidose 3-CBD and CBD proteins in example 1 into PET28a-EGFP vector, purifying by using an escherichia coli gene expression system and an affinity chromatography Ni-NTA column to obtain recombinant proteins, respectively obtaining the Amidose 3-CBD and the CBD with EGFP, naming the two as EGFP-Amidose 3-CBD and EGFP-CBD, and performing SDS-PAGE analysis.
As shown in FIG. 2, the results indicate that the EGFP-Amidase 3-CBD protein was about 60kDa (i.e., the purified product of lane 8 in FIG. 2A), the EGFP-CBD protein was about 40kDa (the purified product of lane 7 in FIG. 2B), the protein sizes on SDS-PAGE gel matched with bioinformatically predicted 64 and 40.1kDa (FIG. 2A and FIG. 2B), and the protein size (about 33 kDa) of the negative control EGFP protein on SDS-PAGE gel matched with bioinformatically predicted (33.2 kDa) (the purified product of lane 8 in FIG. 2C).
Example 3: EGFP-Amidase 3-CBD and EGFP-CBD binding Activity assay
The experimental method comprises the following steps: staphylococcus aureus was grown to mid-log phase at 37℃under shaking culture, then the culture was centrifuged at 12000rpm for 5min, washed once with 50mM Tris-HCl, centrifuged, the bacteria were resuspended with 50mM Tris-HCl and OD was measured 600 Adjust to 0.8. 200 μl OD was taken 600 Mu.l of EGFP-Amidase 3-CBD and EGFP-CBD proteins obtained in example 2 at a concentration of 0.5mg/ml were added to a centrifuge tube, respectively, and incubated at room temperature for 20 minutes. Using 50mM Tris by centrifugation (10000 rpm,3 min)Washing staphylococcus aureus 3 times with HCl to remove unbound protein, re-suspending the washed pellet in 100 μl of 50mm tris-HCl to give a bacterial protein mixed suspension, and detecting by using fluorescence microscopy, flow cytometry, western.
Fluorescence microscopy: mu.l of the final bacterial protein mixed suspension was added to the slide glass, and the slide glass was covered with a cover glass, and the binding effect of the EGFP protein to Staphylococcus aureus was observed (excitation BP 470-490nm; emission: LP 516 nm) under a bright field and FITC filter by an epifluorescence microscope equipped with a U-RFL-T light source (magnification 1000 times), while EGFP protein was prepared as a negative control. To assess the specificity and sensitivity of EGFP-Amidase 3-CBD, staphylococcus aureus strains, escherichia coli O157:157H, salmonella typHimurium, bacillus cereus, propionibacterium acnes, listeria monocytogenes were incubated with the protein, respectively (specific procedure was the same as above) and observed under an epifluorescence microscope, following the same procedure as above, and the results are shown in Table 1.
Table 1: lysis of cell walls of different microorganisms by 80 alpha endolysin, EGFP-amidase 3-CBD
Note that: a ++, has cleavage activity; -, no cleavage activity; b ++, has cell wall binding activity; no cell wall binding activity.
Flow cytometry detection: samples were taken from 50 μl of the final bacterial protein mixture and analyzed using an Accuri C6 Plus flow cytometer equipped with a diode blue laser (excitation wavelength 488 nm) to collect 40000 events. Fluorescence was detected by a 525/50nm bandpass filter on the FL1 channel and data analysis was performed using FlowJo-V10 software.
western detection: mu.l of 5 x SDS loading buffer was pipetted into 20. Mu.l of the final bacterial protein suspension, boiled at 100℃for 5 minutes, electrophoretically analysed using SDS-PAGE pre-gels, and the proteins transferred onto PVDF membranes by transfer techniques and Western blot analysis using anti-histidine antibodies.
The fluorescence microscope detection result shows that the fusion protein EGFP-Amidase 3-CBD and EGFP-CBD can be combined with staphylococcus aureus cells; the observation of fluorescence intensity under a microscope shows that EGFP-Amidase 3-CBD has stronger binding capacity than EGFP-CBD. While negative control EGFP showed no fluorescent cells observed under the fluorescence microscope, see specifically fig. 3A.
As shown in FIG. 3B and FIG. 3C, the detection results of the flow cells showed that the peak value of EGFP-Amidase 3-CBD was significantly shifted to the right than that of the positive control, and the average fluorescence intensity of EGFP-Amidase 3-CBD was greater than that of EGFP-CBD, which had a very significant difference (P < 0.0001), indicating that EGFP-Amidase 3-CBD had a stronger binding capacity than EGFP-CBD, whereas the average fluorescence intensity value of the negative control EGFP also appeared in FIG. 3C, and the analysis was due to the fact that unbound protein was not washed out after protein bound to Staphylococcus aureus, belonging to normal experimental errors.
As shown in FIG. 4, the grayscale value of the EGFP-Amidase 3-CBD band was higher than that of EGFP-CBD, and it was also seen from comparison of the nonspecific bands that the nonspecific bands of EGFP-Amidase 3-CBD were much lighter than that of EGFP-CBD, indicating that EGFP-Amidase 3-CBD had a stronger binding ability than EGFP-CBD.
Example 4: EGFP-amidase 3-CBD protein stability investigation
The EGFP-amidase 3-CBD protein obtained in example 2 was subjected to stability studies at a temperature of 4-60℃and a pH of 3-12 and a NaCl concentration of 0-1000mM, respectively, and the protein treatment time was 30min under each condition, and the amidase3-CBD binding activity to Staphylococcus aureus was measured by using the method of functional analysis of 80. Alpha. Endolysin C-terminus.
Wherein, different pH conditions are realized by buffers with different pH values: glycine-HCl ph=3, sodium acetate ph=5, tris-HCl ph=7, glycine-NaOH ph=9, KCl-NaOH ph=12.
Wherein, under each condition, the protein treatment time was 30min, and the amidase 3-SH3b binding activity to bacteria was measured using the method for measuring in functional analysis of the C-terminus of 80. Alpha. Endolysin.
As shown in FIG. 5, the binding capacity after 100mM NaCl treatment was the strongest, and the EGFP-amidase3-CBD protein was gradually decreased in binding to Staphylococcus aureus with increasing concentration of NaCl, and the fluorescence peak and average fluorescence intensity of the EGFP-amidase3-CBD protein after 100mM NaCl treatment were the largest in the loss cytometer detection. EGFP-amidase3-CBD binding was significantly reduced when NaCl concentration exceeded 200 mM.
As shown in FIGS. 6-7, EGFP-amidase3-CBD protein binding to Staphylococcus aureus was best when the temperature was 4deg.C and pH=7, as shown in the results of EGFP-amidase3-CBD protein treatment at different temperatures and pH.
Example 5: expression and purification of EGFP-RBP
1. Phage 80. Alpha. RBP gene sequences were obtained from NCBI (Genbank accession number ABF 71632.1), mock cloned using snapgene software, and the molecular weight and isoelectric point of the protein calculated using ExPASy. Wherein the amino acid sequence of phage 80 alpha RBP is shown in SEQ ID NO. 13.
2. The gene encoding phage 80 alpha RBP in step 1 is synthesized artificially, and the synthesized gene sequence (DNA sequence) is cloned into a PMV vector to obtain plasmid PMV-RBP. This was cloned into the EGFP-containing pET28 vector by double enzyme digestion (BamHI/XhoI) to produce a pET28a-EGFP-RBP recombinant expression vector. And transforming the recombinant expression vector into E.coli DH5 alpha competent cells by a heat shock method. Then, single colonies were picked up in LB liquid medium containing kanamycin, cultured overnight at 37℃and plasmids were extracted by alkaline lysis and sequenced (sequencing results are shown in FIG. 8), and the correct sequence was determined as a positive transformant.
3. Transformation of the correctly sequenced pET28a-EGFP-RBP from step 2 into E.coli BL21 (DE 3) strain at OD 600 When it is reached to about 0.6,to the culture was added IPTG (isopropyl-. Beta. -d-thiogalactoside) at a final concentration of 1mM, and protein expression was induced at 19℃for 20 hours. For purification, the harvested cells were resuspended in lysis buffer (50 mM Tris-HCl, 500mM NaCl, 5mM imidazole), sonicated for 3min, then centrifuged at 10000rpm for 30min to obtain a soluble protein supernatant, the collected supernatant sample was added to an equilibrated Ni column, the solution flowing out of the Ni column was a permeate, and then the target protein in the permeate was eluted sequentially using 50mM imidazole-containing buffer (wash solution), 250mM imidazole-containing buffer (wash solution 1) and 500mM imidazole-containing eluent (wash solution 2), respectively. After this, the protein was concentrated using a concentration column and buffer was replaced, and the protein was stored in 50mM Tris-HCl. After purification, the proteins were analysed using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) pre-gels. Protein concentration was determined using Bradford protein concentration determination kit, see figure 9 for results.
FIG. 9A shows that the protein size (approximately 33 kDa) of the negative control EGFP protein on SDS-PAGE gel matched the bioinformatically predicted molecular weight (33.2 kDa). FIG. 9B shows that the purified product shows a single band of about 100kDa by SDS-PAGE analysis, matching the bioinformatically predicted molecular weight (104.1 kDa).
The test results show that: successfully expresses and purifies to obtain phage 80 alpha RBP.
Example 6: EGFP-RBP and staphylococcus aureus binding Activity investigation
The binding capacity of EGFP-RBP obtained in example 5 to Staphylococcus aureus was examined by fluorescence microscopy, flow cytometry, western.
The test method comprises the following steps: staphylococcus aureus was cultivated in LB broth at 37℃to log phase, then the culture was centrifuged at 12000rpm for 5min, resuspended in 50mM Tris-HCl and OD 600 Adjust to 0.6. Take 100. Mu.L OD 600 The bacterial solutions of 0.6 were placed in centrifuge tubes, 50. Mu.L of EGFP-RBP protein of 0.5mg/mL was added, and incubated at room temperature for 30min. The bacteria were washed 3 times with 50mM Tris-HCl by centrifugation (10000 rpm,3 min) to remove unbound proteins. Resuspension of the washed pelletFloat in 100. Mu.L of 50mM Tris-HCl.
Fluorescence microscopy: 10. Mu.L of the resuspension was added to the slide and covered with a cover slip. The binding effect of the protein to the bacteria was observed under bright field and FITC filters (excitation BP 470-490nm; emission: LP 516 nm). Bacterial cells supplemented with EGFP protein were also prepared as negative controls. When 80 alpha RBP and green fluorescent protein (EGFP) are fused and expressed, the binding capacity of RBP and staphylococcus aureus can be visually seen through a fluorescence imaging mode.
Flow cytometry detection: samples were analyzed using an Accuri C6 Plus flow cytometer equipped with a diode blue laser (excitation wavelength 488 nm) to collect 10000 events altogether and fluorescence was detected by a 533/30nm fluorescence detector on the FL1 channel. And data analysis was performed using FlowJo-V10 software.
Western Blot detection: mu.L of the resuspended bacteria liquid was pipetted and mixed with 5. Mu.L of 5X SDS Loading buffer and boiled at 100℃for 5 minutes. Electrophoresis analysis was performed using SDS-PAGE pre-gels, proteins were transferred to PVDF membrane by transfer membrane technology, western blot analysis was performed using anti-histidine primary antibodies and HRP-labeled secondary antibodies, and protein bands were visualized using ECL kit and image analyzer.
Fluorescence microscopy showed (FIG. 10A), EGFP-RBP binding to Staphylococcus aureus cells and showing green fluorescence under blue excitation.
The flow cytometry results showed (fig. 10B) that the mean fluorescence intensity of EGFP-RBP was significantly stronger than that of EGFP, with a very significant difference (P < 0.0001).
Western results also showed (FIG. 10C) that the grayscale values of EGFP-RBP bands were higher than those of EGFP.
All three experimental results verify that EGFP-RBP has binding capacity to Staphylococcus aureus.
Example 7: investigation of efficiency of Ni-NTA magnetic beads with binding proteins to capture Staphylococcus aureus
1. Preparation of EGFP-amidase 3-CBD-Ni-NTA magnetic beads (also called EGFP-amidase 3-CBD-Ni magnetic beads)
Ni-polymethacrylate magnetic beads (Ni-NTA magnetic beads, tin-free Bai Mei-Bager Biotechnology Co., ltd.) have Ni-polymethacrylate groups on the surface, ni-polymethacrylate magnetic affinity ligand Ni ions can bind with N-terminal 6 xHis-tag on protein, the N-terminal of EGFP-amidase 3-CBD protein obtained in example 2 is fused with 6 xHis-tag, then 100. Mu.l of EGFP-amidase 3-CBD protein containing His-tag (buffer formulation: 50mM tris-HCl, 100mM NaCl, pH=7.4) and 100. Mu.l of Ni-polymethacrylate magnetic beads are combined by incubation in a mixer for 20min, wherein EGFP-amidase 3-CBD protein is added in excess compared with Ni-polymethacrylate magnetic beads; the Ni-polymethacrylate beads were then separated by a magnetic rack and washed twice with T100. Washing with 50mM Tris-HCl for 2 times, finally re-suspending the magnetic beads in 100 mu l of 50mM Tris-HCl to obtain a magnetic bead suspension, storing at 4 ℃, and observing the coating condition of the Ni-polymethacrylate magnetic beads by adopting a fluorescence microscope, wherein the magnetic beads which are not marked by EGFP-amidase 3-CBD protein have no fluorescence signal observed under a FITC filter, and the magnetic beads marked by the EGFP-amidase 3-CBD protein have strong green fluorescence signal observed under the FITC filter.
2. Preparation of EGFP-RBP-Ni-NTA magnetic beads (also called EGFP-RBP-Ni magnetic beads)
(1) 100. Mu.L of Ni-polymethacrylate beads (Ni-NTA beads, tin-free Baimei biotechnology Co., ltd.) were placed in a 1.5mL EP tube, placed in a magnetic rack, the supernatant was discarded, 500. Mu.L of 50mM Tris-HCl was added to resuspend the beads, and mixed well, placed in a magnetic separation rack, the supernatant was discarded, washed repeatedly 3 times, and finally suspended in 100. Mu.L of 50mM Tris-HCl.
(2) 60 μL of EGFP-RBP (1 mg/mL) protein prepared in example 5, wherein the N-terminal of the EGFP-RBP protein contains a 6 XHis-tag label, is added, and the mixture is manually rotated and mixed for 20min to mark magnetic beads, and the supernatant is discarded.
(3) Adding 500 mu L of 50mM Tris-HCl to resuspend the magnetic beads, uniformly mixing, placing the mixture on a magnetic separation frame for 10s, discarding the supernatant, repeatedly washing for 2 times, finally suspending the mixture in 100 mu L of 50mM Tris-HCl to obtain a magnetic bead suspension, and taking 10 mu L of samples for observation by a fluorescence microscope. The remainder was stored at-4 ℃.
From the results of FIG. 11, it was shown that no fluorescent signal was observed for the beads not labeled with EGFP-RBP protein under the FITC filter, whereas a strong green fluorescent signal was observed for the beads labeled with EGFP-RBP protein under the FITC filter. Further, by observation with a fluorescence microscope, the inventors surprisingly observed that EGFP-RBP protein uniformly covered the surface of Ni-NTA magnetic beads. And when EGFP-RBP is added, the observed green fluorescence is in one-to-one comparison with the position of the bright-field staphylococcus aureus when the peak value of the excitation light is 488 nm.
The above results show that the EGFP-RBP protein obtained by the invention can easily realize the uniform coating of the surface of the Ni-polymethacrylate magnetic beads, and the EGFP-RBP-Ni magnetic beads (also called EGFP-RBP-Ni-NTA magnetic beads) are successfully prepared.
3. The above obtained EGFP-RBP protein-coated Ni-polymethacrylate magnetic beads (EGFP-RBP-Ni-NTA magnetic beads) and EGFP-amidase3-CBD protein-coated Ni-polymethacrylate magnetic beads (EGFP-amidase 3-CBD-Ni-NTA magnetic beads) were subjected to detection of the capture efficiency of staphylococcus aureus, and the staphylococcus aureus-captured magnetic beads were subjected to plate culture and counting, and the specific procedures were as follows:
1ml of Staphylococcus aureus (10) 1 -10 4 CFU/ml) was incubated with 100 μl of the bead suspension on a mixer for 40min, the beads were separated by a magnetic rack, washed 2 times with 50mM Tris-HCl, finally the beads were resuspended in 1ml of 50mM Tris-HCl, 1ml of the bead suspension was directly plated, after incubation for 24h at 37 ℃, cells were counted by colony, and the results of EGFP-amidase 3-CBD-Ni-NTA beads are shown in fig. 12 to 13, and those of EGFP-RBP-Ni-NTA beads are shown in fig. 14. Wherein, magnetic bead capturing efficiency (100%) = (number of magnetic bead capturing/number of supernatant residue+number of magnetic bead capturing) ×100%.
As shown in FIG. 12, the inventors surprisingly found that EGFP-amidase3-CBD protein was very uniformly coated on the surface of Ni-polymethacrylate beads, and that the positions of green fluorescence, red fluorescence and bright field staphylococcus aureus were in one-to-one comparison when the peak of excitation light was 488nm and 532nm, as observed after RFP-amidase 3-CBD was added. The above results show that the EGFP-amidase3-CBD protein obtained by the invention can easily realize even coating of the surface of the Ni-polymethacrylate magnetic beads.
As shown in FIG. 13, the EGFP-amidase 3-CBD protein coated Ni-polymethacrylate beads were loaded with Staphylococcus aureus bioburden (bioberden) at 1X 10 4 The capture efficiency at CFU/ml was 25%; as the bacterial load gradually decreases, the capture efficiency of the magnetic beads is also continuously increased and is 1 multiplied by 10 1 When CFU/ml, the capturing efficiency of the magnetic beads reaches 78%, and a good capturing effect can be achieved.
As shown in FIG. 14, when the number of Staphylococcus aureus was 1X 10 4 During CFU, the number of EGFP-RBP-Ni-NTA magnetic beads is insufficient to capture all staphylococcus aureus, and the capture efficiency of the magnetic beads is continuously increased at 1 multiplied by 10 along with the reduction of staphylococcus aureus 1 At CFU/ml, the number of beads is sufficient for bacteria, and the capturing efficiency of the beads reaches 82%, so that the efficiency of recovering staphylococcus aureus is also increased.
Further, the inventor optimizes the capturing condition of the EGFP-RBP-Ni-NTA magnetic beads to staphylococcus aureus.
4. Optimization of EGFP-RBP-Ni-NTA magnetic bead capturing conditions
(1) EGFP-RBP protein concentration
The concentration of EGFP-RBP directly influences the labeling efficiency of the magnetic beads, and further influences the capturing rate of target bacteria. The experiment was performed by labeling the beads with EGFP-RBP protein concentrations of 10ng, 60ng, 100ng, respectively, and gradient diluting Staphylococcus aureus to about 1X 10 with 50mM Tris-HCl 3 CFU/mL. And (3) performing an experiment according to the step (3), calculating the magnetic bead capturing efficiency, and further determining the optimal EGFP-RBP protein concentration.
From FIG. 15A, it is understood that when the amount of EGFP-RBP protein added was 10ng of the labeled beads, the capturing efficiency of EGFP-RBP-Ni-NTA beads was 50%, and the recovery rate of the beads increased correspondingly with the increase of the amount of EGFP-RBP protein added, but when the amount of protein added reached 100ng, the recovery rate of the beads was 65%, and there was no significant difference in capturing rate (P > 0.05) from the amount of protein added of 60ng, probably because the amount of 60ng of EGFP-RBP protein added had already made the beads saturated. Therefore, an amount of 60ng EGFP-RBP protein was selected for the next experiment.
(2) Number of Ni-NTA magnetic beads
The experiment uses 50mM Tris-HCl to dilute Staphylococcus aureus to about 1X 10 3 CFU/mL, optional addition of 6X 10 4 、7.2×10 4 、9×10 4 、1.2×10 5 Coupling and capturing experiments are carried out on EGFP-RBP-Ni-NTA magnetic beads with different numbers (specific steps refer to step 3 of the embodiment), and the capturing rate is calculated according to the colony number of the flat plate, so that the optimal number of the Ni-polymethacrylate magnetic beads is determined.
As shown in FIG. 15B, with the increasing number of Ni-polymethacrylate beads, the capture rate of the beads was correspondingly increased at 1.2X10 5 The recovery rate reaches 60% when the number of the magnetic beads is counted, but the correspondence is not always present, and too many magnetic beads may cause a decrease in capturing efficiency of target bacteria and too many magnetic beads may not be effectively dispersed when captured. Thus, 1.2X10 were selected for EGFP-RBP-Ni-NTA magnetic beads 5 The following experiment was performed with respect to the number of magnetic beads.
Example 8: investigation of Staphylococcus aureus genome extraction by different extraction methods
1. The steps of extracting staphylococcus aureus genome DNA by an endolysin + water boiling method are as follows:
the 80 alpha Endolysin gene is analyzed and found from phage 80 alpha genome, synthesized from Huada genes and cloned in a PMV vector, the 80 alpha Endolysin gene is cloned in a PET28a vector by a double enzyme digestion (BamHI/XhoI) mode, and the constructed PET28a-Endolysin is transformed into BL21 in OD 600 1mM IPTG was added at=0.6 and induction was performed at 19℃for 20h.10000RPM,5min collecting induced bacteria, suspending the bacteria in lysis buffer (50 mM Tris-HCl, 500mM NaCl, 5mM imidazole), sonicating for 3min, centrifuging at 10000rpm for 30min to obtain soluble protein supernatant, purifying by EGFP-amidase 3-CBD-Ni-NTA magnetic beads obtained in example 2 to obtain target protein, and concentrating the target protein in a buffer solution After this, the protein was concentrated using Amicon Ultra-4centrifugal filters Ultracel-30K and the elution buffer (50 mM Tris-HCl, 500mM imidazole, 100mM NaCl) was replaced, and the protein (80. Alpha. Endolysin protein having the amino acid sequence shown in SEQ ID NO: 15) was stored in 50mM Tris-HCl. After purification, the protein was analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) pre-gel, as shown in FIG. 16A, and the purified 80. Alpha. Endolysin protein showed about 57kDa (i.e., as the purified product of lane 9 in FIG. 16A) on SDS-PAGE analysis, matching the bioinformatically predicted size of the 80. Alpha. Endolysin protein (57.3 kDa). Thus, 80. Alpha. Endolysin (Endolysin) is obtained.
1mL OD 600 Staphylococcus aureus bacterial liquid (staphylococcus aureus ATCC 29213) of which the ratio is=0.8 is placed in a centrifuge tube and centrifuged for 3min at 10000rpm, and the supernatant is discarded; bacteria were suspended by adding 100 μl Tris-HCl (ph=7.4) buffer; adding 40 mu l of 400 mu g/ml 80 alpha endolysin protein obtained above, and reacting for 30min; the supernatant was aspirated for qPCR by centrifugation 10000r in a boiling water bath for 10min, 3min and centrifugation.
Wherein, qPCR system is as follows:
| genomic DNA | Total volume of 10. Mu.l |
| 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 |
| H 2 O | 3.4μl |
The circulation system is as follows: 95 ℃ for 15min;95 ℃, 15s,60 ℃, 20s,30 cycles, 72 ℃ and 45s.
2. The steps of extracting staphylococcus aureus genome DNA by the water boiling method are as follows:
1mL OD 600 Staphylococcus aureus bacterial liquid (staphylococcus aureus ATCC 29213) of which the ratio is=0.8 is placed in a centrifuge tube and centrifuged for 3min at 10000rpm, and the supernatant is discarded; bacteria were suspended by adding 140 μl Tris-HCl (ph=7.4) buffer; water bath at 100deg.C for 10min, centrifuging 10000r,3min, and absorbing supernatant for qPCR.
Wherein, qPCR system is as follows:
| genomic DNA | Total volume of 10. Mu.l |
| 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 |
| H 2 O | 3.4μl |
The circulation system is as follows: 95 ℃ for 15min;95 ℃, 15s,60 ℃, 20s, 30 cycles, 72 ℃ and 45s.
As shown in FIG. 16B, in the experiment of the lysis of Staphylococcus aureus ATCC29213 with endolysin protein (concentration of 400. Mu.g/ml), 80. Alpha. Endolysin can reduce the absorbance at 600nm from 1.0 to 0.2 in 60 minutes at 400. Mu.g/ml, and the result shows that the endolysin protein has good lysis activity.
As shown in fig. 16C and 16D, when Ct Threshold in QPCR is set to 2000, the Ct values of endolysin+water boiling method and water boiling method are 10 and 15, respectively, which indicates that the extraction yield of staphylococcus aureus genomic DNA of endolysin+water boiling method is far higher than that of water boiling method, which is because staphylococcus aureus cell wall is thicker, normal water boiling method cannot effectively break staphylococcus aureus cell wall, thus resulting in low genome DNA efficiency, whereas endolysin is phage-encoded peptidoglycan hydrolase, which can specifically lyse host bacterial cell wall, thus resulting in release of intracellular substances, when endolysin is combined with water boiling method, the extraction yield is much higher than that of water boiling method alone, thus providing a very important effect for subsequent rapid detection.
Example 9: specific fragment genome nuc fragment amplification verification of staphylococcus aureus
The procedure for amplifying the nuc fragment of the staphylococcus aureus genome using the RPA technique is as follows.
Preparation of EGFP-amidase 3-CBD-Ni magnetic beads
1. 100. Mu.l of the mixed Ni-polymethacrylate beads were pipetted into an EP tube and washed 2 times with 500. Mu. l T100 for 2min each, the supernatant was discarded and suspended in 100. Mu.l of T100.
2. Mu.l EGFP-amidase 3-CBD protein at a concentration of 1mg/ml was added and incubated for 20min, and the EP tube was tilted 50℃every 2min, gently swirled by hand.
3. Placing the incubated EGFP-amidase 3-CBD protein suspension on a magnetic rack, and standing to remove supernatant. Mu.l of 50mM Tris-HCl was washed 2 times and finally suspended in 100. Mu.l of 50mM Tris-HCl.
(II) capturing staphylococcus aureus by using EGFP-amidase 3-CBD-Ni magnetic beads
4. Centrifuging 3ml of Staphylococcus aureus, washing with 1ml of Tris-HCl once, and adjusting to OD with Tris-HCl 600 =0.6, re-diluted to 1×10 4 CFU/ml、1×10 3 CFU/ml、1×10 2 CFU/ml、1×10 1 CFU/ml, four dilutions of bacterial solution were obtained.
5. Taking 1ml of the four dilutions of bacterial solutions respectively, and incubating for 20min in EGFP-amidase 3-CBD protein suspension, wherein the EP tube is inclined for 50 degrees every 2min, and slightly rotated and rocked by hands.
6. The incubated EGFP-amidase 3-CBD protein-Staphylococcus aureus, the suspension was placed on a magnetic rack, the supernatant was discarded, and 500. Mu.l of 50mM Tris-HCl was added for 2 washes, 2min each time, and resuspended in 40. Mu.l of 50mM Tris-HCl.
Extraction of staphylococcus aureus DNA
7. 10 μl of 2mg/ml endolysin protein (see step 1 of example 8 specifically) was added, incubated for 30min, then placed in a boiling water bath for 10min, and the supernatant was aspirated to give a crude extract.
Amplification of nuc fragment of staphylococcus aureus genome
8. RPA is carried out on the crude extract, and the specific steps are as follows:
the Primer used in the RPA reaction was designed by Primer Premier 5.0, and the Primer length was between 30nt and 35 nt.
Wherein RPA-1-F:5'-GCATCACAAACAGATAACGGCGTAAATAGAAG-3' (SQE ID NO: 3);
RPA-1-R：5’-ACATTAATTTAACCGTATCACCATCAATCGCT-3’(SQE ID NO:4)；
RPA-2-F：5’-CATCACAAACAGGTAACGGCGTAAATAGAAGT-3’(SQE ID NO:5)；
RPA-2-R：5’-TCTCTACACCTTTTTTAGGATGCTTTGTTTCA-3’(SQE ID NO:6)。
the reaction system of RPA is: 10. Mu.M of the upstream primer (RPA-1-F or RPA-2-F) and 10. Mu.M of the downstream primer (RPA-1-R or RPA-2-R), 50mM Tris-HCl (pH 7.5), 100mM potassium acetate, 14mM magnesium acetate, 2mM Dithiothreitol (DTT), 5% polyethylene glycol, 200. Mu.M dNTP, 3mM ATP, 1. Mu.l of 5000units/ml Bsu DNA polymerase and 2. Mu.l of amplified product. The mixture was incubated in a conventional water bath at 37℃to perform the RPA reaction, wherein the concentrations of the respective substances in the mixture were all the final concentrations, RPA-1-F and RPA-1-R were amplified to give nuc fragment 1, and RPA-2-F and RPA-2-R were amplified to give nuc fragment 2.
The experimental method for amplifying the escherichia coli genome nuc fragment by using the RPA is the same as the experimental method for amplifying the staphylococcus aureus genome nuc fragment by using the RPA, and the difference is only that the selected bacterial liquid is different.
The amplification products of Staphylococcus aureus and Escherichia coli were extracted with phenol/chloroform, respectively, and subjected to 1% agarose gel electrophoresis, and the results are shown in FIG. 17.
The results in FIG. 17 show that clear bands are seen in the Staphylococcus aureus lane, while no bands are seen in the E.coli lane, indicating that the nuc gene is a Staphylococcus aureus-specific gene and can be amplified by isothermal RPA amplification.
As shown in FIG. 18, for the sensitivity of RPA, isothermal amplification of RPA was performed using 10-fold serial dilutions of Staphylococcus aureus genomic DNA as a template, and showed a lower detection limit of 10 on agarose gel electrophoresis 2 aM。
The experimental results show that the staphylococcus aureus specific nuc gene fragment is successfully amplified in the embodiment.
Example 10: detection of Staphylococcus aureus using EGFP-RBP-Ni-NTA magnetic beads or EGFP-amidase 3-CBD-Ni magnetic beads
1. Specificity investigation of EGFP-amidase 3-CBD-Ni magnetic bead-Recombinase Polymerase Amplification (RPA) detection method for staphylococcus aureus
The EGFP-amidase 3-CBD-Ni magnetic beads obtained in example 2 were used to capture and extract the genomic DNA of Staphylococcus aureus in a sample, and the sample was 1X 10 4 CFU/ml Staphylococcus aureus contaminated Tris-HCl, milk, orange juice and cheese, bound and captured using EGFP-amidase 3-CBD-Ni magnetic beads obtained in example 2Staphylococcus aureus, genomic DNA was extracted using an endolysin + water-boiling method followed by isothermal amplification of nuc fragments using RPA (see in particular example 9) and running the gel on a 1% agarose gel as shown in fig. 19.
The results showed that contaminated Tris-HCl, milk, orange juice and cheese all successfully amplified nuc gene fragment and the brightness of the bands was almost identical.
The test results show that the magnetic beads capture staphylococcus aureus and are not influenced by food matrixes.
2. Specific investigation of EGFP-RBP-Ni-NTA magnetic bead combined PCR method for detecting staphylococcus aureus
2.1 1mL of 1X 10 7 CFU/mL Staphylococcus aureus and Escherichia coli, 1.2X10 obtained in example 5 was added 5 Enrichment isolation of EGFP-RBP-Ni magnetic beads (see step 3 of example 7) was performed and finally resuspended in 50. Mu.L of 50mM Tris-HCl. The genomic DNA was extracted by the water boiling method (boiling water treatment for 10min, see step 1 of example 8). And amplifying the staphylococcus aureus specific gene nuc by using the extracted genome DNA as a template in a PCR mode, so as to verify the specificity of the EGFP-RBP-Ni magnetic beads for capturing staphylococcus aureus.
Wherein, the primer of PCR is Nuc R: CGTATTGCCCTTTCGAAACATT (SEQ ID NO: 2), nuc F: GGCATATGTATGGCAATTGTTTC (SEQ ID NO: 1). The PCR system is shown in the following table:
forward and reverse primers were each 1ul, pre-denatured for 5min at 98 ℃, denatured for 10s at 98 ℃, annealed for 5s at 60 ℃ and extended for 10s at 72 ℃ for 30 cycles. Extension was terminated at 72℃for 5min and at 4 ℃.
As shown in FIG. 20A, lane 1 is the nuc gene fragment product amplified by combining the EGFP-RBP-Ni-NTA magnetic beads with PCR reaction conditions after capturing Staphylococcus aureus.
The results show that: the EGFP-RBP-Ni magnetic beads can effectively enrich and separate staphylococcus aureus, and can be further used for detecting staphylococcus aureus in a sample to be detected by a PCR method.
2.2 sensitivity investigation of method for detecting staphylococcus aureus by combining EGFP-RBP-Ni magnetic beads with PCR method
1.2X10 obtained in example 5 was taken 5 EGFP-RBP-Ni-NTA magnetic beads were added at 1mL of each concentration (0, 1X 10) 3 、1×10 4 、1×10 5 、1×10 6 、1×10 7 CFU/mL) of Staphylococcus aureus was isolated for enrichment (see, in particular, step 3 of example 7) and resuspended in 50. Mu.L of 50mM Tris-HCl. The genomic DNA was extracted by the water boiling method (boiling water treatment for 10min, see step 1 of example 8). The extracted genome DNA is used as a template, a PCR method is adopted to amplify specific nuc gene fragments of staphylococcus aureus, and the sensitivity of the EGFP-RBP-Ni magnetic bead detection method of staphylococcus aureus is examined.
As a result, as shown in FIG. 20B, when the amount of EGFP-RBP-Ni-NTA magnetic beads was 1.2X10 5 When the sample to be detected is 1mL, the lowest detection concentration of staphylococcus aureus in the sample to be detected is 10 3 CFU/mL. The experimental results show that the method provided by the invention has better sensitivity.
2.3 application of magnetic bead capture staphylococcus aureus in actual food sample quality detection
The main challenge is whether the food can be widely applied in different food environments. Therefore, the inventor pollutes the staphylococcus aureus with Tris-HCl, milk and orange juice to ensure that the pollution concentration of the staphylococcus aureus is 1 multiplied by 10 3 CFU/mL, after capturing staphylococcus aureus in contaminated food samples by using EGFP-RBP-Ni-NTA magnetic beads prepared in example 5, extracting genome DNA by using a water boiling method, amplifying nuc gene fragments specific to staphylococcus aureus by using PCR, and running gel by using 1% agarose gel electrophoresis according to the method of step 2.2 in the example.
As shown in FIG. 21, tris-HCl, milk, orange juice contaminated with Staphylococcus aureus amplified nuc gene fragment, and the band brightness of each sample was substantially unchanged. Shows that the EGFP-RBP-Ni-NTA magnetic beads can capture staphylococcus aureus and are not influenced by food matrixes, Can be widely applied to quality detection of staphylococcus aureus in food, and the detection limit is 1 multiplied by 10 3 CFU/mL。
Example 11: detection of staphylococcus aureus in milk by combining magnetic bead capture and cas12a/crRNA cleavage
1. Three different crrnas were selected (see fig. 23A) and nuc fragment 1 and nuc fragment 2 obtained in example 9 were subjected to cas12a/crRNA cleavage assay, and cas12a/crRNA cleavage assay methods were as follows:
500nM crRNA, 250nM Cas12a, 2.5. Mu.M ssDNA-FQ reporter (5 '-FAM-TTATT-BHQ-1-3'), 2. Mu.L NEB buffer 3.1, 3. Mu.L of the amplified product obtained in example 7 were mixed and ddH was added 2 O to 20. Mu.L. The reaction was performed at 37℃and on a qPCR machine for 30min, with fluorescence measurements taken every 1min, see in particular FIGS. 22-23. Wherein crRNA-1 is used to recognize nuc fragment 1, crRNA-2 and crRNA-3 are used to recognize nuc fragment 2, crRNA is as follows:
as shown in FIG. 23C, crRNA-1 activated cas12a/crRNA cleaved ssDNA-FQ reporter resulted in the strongest fluorescence, thus indicating that crRNA-1 is the optimal choice of three crRNAs. As shown in FIG. 23B, it can be seen that the fluorescence value can be generated only when all the solvents in the reaction system are added in the different component analyses of cas12a/crRNA cleavage reaction, which is indispensable.
2. In order to rapidly detect staphylococcus aureus in food, prevent occurrence of food-borne diseases caused by staphylococcus aureus, staphylococcus aureus was captured by using the upstream EGFP-amidase 3-CBD-Ni magnetic beads obtained in example 2, and combined with downstream RPA, cas12a cleavage detection, whether food was contaminated was observed and evaluated by naked eyes under irradiation of ultraviolet light, and the specificity and sensitivity of the method were examined.
The test method comprises the following steps: 500nM crRNA-1 (see step 1 of this example specifically), 250nM Cas12a, 2.5. Mu.M ssDNA-FQ reporter (5 '-FAM-TTATT-BHQ-1-3') 2. Mu.L NEB buffer 3.1, 3. Mu.L of the RPA amplification product obtained in example 9 (containing the yellow staphylococcal genomic nuc fragment) were mixed and ddH was added 2 O to 20. Mu.L. The reaction was performed at 37℃and on a qPCR machine for 30min, with fluorescence measurements taken every 1 min.
Specificity investigation and results: selecting 1×10 4 The method in example 9 was used to capture staphylococcus aureus in samples, extract staphylococcus aureus genomic DNA and RPA-amplified staphylococcus aureus genomic nuc fragments, and detect staphylococcus aureus with cas12a/crRNA cleavage to detect if specific.
As shown in FIG. 24, staphylococcus aureus activated cas12a and cleaved ssDNA-FQ reporter to generate fluorescence values.
The experimental result shows that the EGFP-amidase 3-CBD-Ni magnetic beads capture staphylococcus aureus and can specifically detect staphylococcus aureus in food by combining with cas12a/crRNA cleavage detection method.
Sensitivity investigation and results: respectively 1×10 3 CFU/ml、1×10 2 CFU/ml、1×10 1 CFU/ml staphylococcus aureus contaminated sterile milk, then adding 100 mu l EGFP-amidase 3-CBD-Ni magnetic beads, extracting staphylococcus aureus genome DNA by using an endolysin + water boiling method, performing RPA reaction at 37 ℃ for 30min (see example 9 in particular), and performing Cas12a cleavage at 37 ℃ for 30min. Under the irradiation of ultraviolet light, the fluorescent signal is checked by naked eyes; and analyzing the data using GraphPad Prism 8.0 software and presenting with mean ± standard deviation, wherein the differences between the data means are analyzed by t-test, P<At 0.05, the difference was significant.
As shown in FIG. 25, FIG. A, B, C is a method of capturing and binding cas12a/crRNA cleavage using EGFP-amidase 3-CBD-Ni magnetic beads, respectivelyDetection of 1X 10 in milk 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 cleavage reaction in a QPCR instrument set at 37℃for 30min, and the tube plot in the curve is the corresponding fluorescence plot under UV irradiation after cleavage at 37℃for 30min.
The results show that the lowest detection limit of EGFP-amidase 3-CBD-Ni magnetic bead capture combined cas12a/crRNA cleavage detection can be further reduced to 1X 10 1 CFU/ml。
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (10)
1. An adsorptive magnetic bead comprising:
ni-polymethacrylate magnetic beads;
and the binding protein is used for binding staphylococcus aureus, and the Ni-polymethacrylate magnetic beads are connected with the binding protein.
2. The adsorptive magnetic bead of claim 1, wherein the N-terminus of said binding protein is linked to said Ni-polymethacrylate magnetic bead;
optionally, the binding protein is derived from a staphylococcus aureus bacteriophage, preferably from staphylococcus aureus bacteriophage 80 a;
optionally, the binding protein comprises at least one of a receptor binding protein derived from a staphylococcus aureus phage and an Amidase protein;
preferably, the binding protein comprises at least one selected from the group consisting of a receptor binding protein derived from staphylococcus aureus phage 80 a and an Amidase protein;
optionally, the Amidase protein is an Amidase 3-CBD protein;
optionally, the receptor binding protein has at least one of the amino acid sequence shown as SEQ ID NO. 13, an amino acid sequence having at least 90% homology with SEQ ID NO. 13, and an amino acid sequence having equal or similar Staphylococcus aureus binding properties to the receptor binding protein shown as SEQ ID NO. 13;
Optionally, the Amidase 3CBD protein has an amino acid sequence shown as SEQ ID NO. 14, an amino acid sequence with at least 90% homology with SEQ ID NO. 14, and an amino acid sequence with equal or similar staphylococcus aureus binding performance to the Amidase 3CBD protein shown as SEQ ID NO. 14;
optionally, the binding protein has a fluorescent group attached;
preferably, the fluorescent group is an EGFP group;
optionally, further comprising a protein tag;
optionally, the C-terminus of the protein tag is linked to the N-terminus of the binding protein, and the N-terminus of the protein tag is linked to the Ni-polymethacrylate magnetic beads;
optionally, the protein tag comprises at least one selected from the group consisting of a His tag, a Flag tag, a GST tag, an MBP tag, a SUMO tag, and a C-Myc tag;
preferably, the protein tag is a His tag.
3. Use of the magnetic adsorption beads of any one of claims 1-2 in the preparation of a kit for detecting staphylococcus aureus.
4. A kit, comprising:
the adsorption magnetic bead according to any one of claims 1 to 2;
optionally, further comprising:
at least one of Cas12a, crRNA, and ssDNA-FQ reporter;
Preferably, cas12a, crRNA, and ssDNA-FQ reporter are further included.
5. Use of the magnetic adsorption beads of any one of claims 1-2 or the kit of claim 4 in the manufacture of a product for detecting staphylococcus aureus.
6. A method of detecting staphylococcus aureus, comprising:
the adsorption magnetic beads according to any one of claims 1 to 2 or the kit according to claim 4 are used for carrying out contact treatment on a sample to be tested so as to detect whether staphylococcus aureus or the content of staphylococcus aureus is contained in the sample to be tested.
7. The method as recited in claim 6, further comprising:
carrying out DNA extraction treatment on the contact treatment product;
amplifying the DNA extraction treatment product;
detecting an amplification treatment product so as to detect whether staphylococcus aureus or the content of the staphylococcus aureus is contained in the sample to be detected;
optionally, the adsorption magnetic beads are obtained by: carrying out first mixing treatment on Ni-polymethacrylate magnetic beads and binding proteins;
optionally, the binding protein is Amidase 3-CBD protein, and the first mixing treatment is carried out at a temperature of (2-30) DEG C and a pH value of 5-9;
Optionally, the binding protein is an Amidase 3-CBD protein, and before the contacting treatment, naCl and the adsorption beads are subjected to a second mixing treatment;
optionally, the final concentration of NaCl is 50-200 mM;
optionally, the number of the adsorption magnetic beads is not less than 1.0X10 5 And each.
8. The method of claim 7, wherein the DNA extraction process comprises:
boiling the contact treatment product and endolysin in water;
optionally, before the poaching treatment, reacting the endolysin with the contact treatment product for 20-40min;
optionally, the time of the water boiling treatment is 5-20min;
optionally, the volume ratio of the endolysin to the sample to be tested is 1: (40-60), wherein the concentration of endolysin is 1-3mg/ml;
optionally, the endolysin is selected from phage endolysins derived from the staphylococcus aureus;
optionally, the amplification treatment is performed using a recombinase polymerase amplification method.
9. The method of claim 7, wherein the detecting comprises:
performing third mixing treatment on the amplification treatment product, the Cas12a, the crRNA and the ssDNA-FQ reporter;
Based on the fluorescence signal generated by the third mixing process, detecting whether staphylococcus aureus or the content of the staphylococcus aureus is contained in the sample to be detected;
optionally, the third mixing process produces a fluorescent signal that is indicative of the presence of staphylococcus aureus in the sample to be tested; or alternatively
The third mixing process does not generate a fluorescent signal, and is an indication that the sample to be tested does not contain staphylococcus aureus;
optionally, the detecting further comprises:
and determining the content of staphylococcus aureus in the sample to be detected based on a standard curve, wherein the standard curve is a curve corresponding to the preset quantity of staphylococcus aureus and fluorescence signal intensity.
10. The method of claim 9, wherein the crRNA has a nucleotide sequence as set forth in SEQ ID No. 7 or a nucleotide sequence having at least 90% homology thereto.
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