CN114317787A - Adsorption magnetic beads, kit and use thereof, and method for detecting Staphylococcus aureus - Google Patents

Abstract

The invention relates to an adsorption magnetic bead and a kit and application thereof as well as a method for detecting Staphylococcus aureus. The adsorption magnetic beads are selected from Ni-polymethacrylate magnetic beads, and the Ni-polymethacrylate magnetic beads are linked to the Amidase 3-CBD protein through a His-tag sequence. The adsorption magnetic beads can specifically bind to Staphylococcus aureus, and capture the Staphylococcus aureus in the sample, so as to facilitate subsequent acquisition and/or detection of the Staphylococcus aureus in the sample.

Description

Adsorption magnetic beads, kit and use thereof, and method for detecting Staphylococcus aureus

technical field

The invention belongs to the technical field of biological detection, in particular to an adsorption magnetic bead, a kit and application thereof, and a method for detecting Staphylococcus aureus.

Background technique

In recent years, outbreaks of foodborne diseases caused by eating food contaminated with harmful bacteria have increased dramatically, and detection of harmful bacteria in food has become an important factor related to preventing food safety and protecting public health. Staphylococcus aureus is one of the most common food-borne pathogens, which can produce enterotoxins and cause food poisoning. Food poisoning has always been a serious problem plaguing some remote areas, and traditional methods, such as plate counting and polymerase chain reaction, are time-consuming and instrument-dependent, severely limiting the rapid detection of food-borne pathogens in remote areas.

Therefore, there is a need to develop products or methods for rapid detection and convenient use.

SUMMARY OF THE INVENTION

The present invention aims to solve at least one of the technical problems existing in the prior art at least to a certain extent. To this end, the present invention provides an adsorption magnetic bead and a kit and application thereof as well as a method for detecting Staphylococcus aureus, the adsorption magnetic beads can specifically bind to Staphylococcus aureus and capture Staphylococcus aureus in a sample , to facilitate subsequent acquisition and/or detection of Staphylococcus aureus in samples.

The present invention is accomplished based on the following findings of the inventors:

Several methods have been developed for the rapid detection and identification of S. aureus in food samples, such as polymerase chain reaction (PCR), enzyme-linked immunosensors, and nucleic acid-based molecular biology methods. These advanced microbial detection methods will detect The time has been shortened from days to hours, but there are still some limitations, for example, these methods are often expensive, require high-tech laboratory equipment and skilled technicians, and cannot be used as field detection methods. In addition, in remote areas, the lack of experimental equipment severely limits the detection of foodborne diseases.

Bacteriophage is a host-specific and lytic bacterial virus, endolysins are bacterial cell wall peptidoglycan hydrolases synthesized late in the phage lysis cycle, mediating the lysis of host cells and the release of progeny phages, usually composed of two Modular domain: composed of N-terminal catalytic domain (EAD) and C-terminal cell wall binding domain (CBD). The CBD of endolysin is responsible for attaching hydrolases to specific substrates in bacterial cell walls through non-covalent binding of carbohydrate ligands. It has been reported that the number of CBD binding sites on a bacterial cell can reach ¹⁰⁷ or more.

Based on this, the inventors used Ni-polymethacrylate magnetic beads with Amidase 3-CBD protein, which can bind to Staphylococcus aureus in the sample to be tested, so as to achieve the purpose of capturing Staphylococcus aureus, which is relatively The traditional detection method can reduce the steps of amplifying and culturing Staphylococcus aureus in the sample to be tested, and has the advantages of simple operation and saving detection time. Further, the endolysin (endolysin) of the above-mentioned culture solution and Staphylococcus aureus bacteriophage is extracted by boiling the genome of Staphylococcus aureus in the culture solution, and the endolysin can specifically crack the Staphylococcus aureus cell wall. , resulting in the release of Staphylococcus aureus intracellular substances, which can greatly improve the extraction of the Staphylococcus aureus genome. The extracted genome is then amplified and identified by recombinase polymerase amplification (RPA) and cas12/crRNA cleavage mechanism, and identification can be achieved by only using a UV lamp irradiator. ^The detection ^of Staphylococcus aureus by the above method has strong specificity and high sensitivity. It has the advantages of detecting food-borne pathogens and other advantages. The detection of Staphylococcus aureus by this method does not rely on experimental instruments, which is of great significance for the control of food-borne disease outbreaks.

In one aspect of the present invention, the present invention provides an adsorption magnetic bead. According to an embodiment of the present invention, the adsorption magnetic beads are selected from Ni-polymethacrylate magnetic beads, and the Ni-polymethacrylate magnetic beads are linked to Amidase 3-CBD protein through a His-tag sequence.

After a lot of experiments, the inventor found that Ni ions of Ni-polymethacrylate magnetic beads can be combined with the His-tag sequence of Amidase 3-CBD protein to form Ni-polymethyl methacrylate with Amidase 3-CBD protein. Acrylate-based magnetic beads are adsorption magnetic beads. And the inventor also found that the Amidase 3-CBD protein in the adsorption magnetic beads can specifically bind to Staphylococcus aureus, and when the adsorption magnetic beads are added to the sample, it can specifically capture Staphylococcus aureus, so as to achieve golden yellow color. The capture and enrichment of Staphylococcus is helpful for its subsequent detection, reducing the steps of bacterial culture, shortening the detection time, and improving the detection efficiency. Moreover, the inventor found through experiments that the capture effect of Amidase 3-CBD protein on Staphylococcus aureus was better than that of CBD protein on Staphylococcus aureus.

According to the embodiment of the present invention, the Amidase 3-CBD protein is connected with a fluorescent group, preferably an EGFP group. Thereby, the tracking of the protein is facilitated.

In yet another aspect of the present invention, the present invention provides a kit. According to an embodiment of the present invention, the kit includes: Ni-polymethacrylate magnetic beads; Amidase 3-CBD protein with His-tag sequence.

After a lot of experiments, the inventor found that Amidase 3-CBD protein can specifically bind to Staphylococcus aureus, and the Ni-NTA magnetic beads in the kit were contacted with Amidase 3-CBD protein with His-tag sequence. The Ni ion based on acrylate magnetic beads can be combined with the His-tag sequence of Amidase 3-CBD protein to form Ni-polymethacrylate magnetic beads with Amidase 3-CBD protein. The adsorption magnetic beads can specifically capture Staphylococcus aureus, so as to separate and obtain Staphylococcus aureus, which is helpful for further detection of Staphylococcus aureus and improves detection efficiency. Moreover, the inventor found through experiments that the capture effect of Amidase 3-CBD protein on Staphylococcus aureus was better than that of CBD protein on Staphylococcus aureus.

According to the embodiment of the present invention, the Amidase 3-CBD protein is connected with a fluorescent group, preferably an EGFP group. Thereby, the tracking of the protein is facilitated.

According to the embodiment of the present invention, the Ni-polymethacrylate magnetic beads and the Amidase 3-CBD protein are provided in a manner of combining Ni and His-tag sequence.

It should be noted that the ways of providing Ni-polymethacrylate magnetic beads and Amidase 3-CBD protein with His-tag sequence in the kit of the present invention are not strictly limited, and they can be provided separately. The two are contacted to realize the combination of the two; it can also be directly provided in the form of a conjugate, that is, Ni-polymethacrylate magnetic beads and Amidase 3-CBD protein are combined with Ni and His-tag tag sequences , that is, it is provided in the form of the aforementioned adsorption magnetic beads. When ready to use, the conjugate is directly contacted with the sample to achieve the capture purpose.

In another aspect of the present invention, the present invention proposes the application of the above-mentioned adsorption magnetic beads or the above-mentioned kit in the detection of Staphylococcus aureus. As mentioned above, the use of the aforementioned adsorption magnetic beads or kits can specifically bind to Staphylococcus aureus, thereby realizing the capture and enrichment of Staphylococcus aureus, so there is no need to culture them, simplifying the detection steps and time, and improving the detection efficiency.

In another aspect of the present invention, the present invention provides a method for detecting Staphylococcus aureus. According to an embodiment of the present invention, the method includes: step 1: using the above-mentioned adsorption magnetic beads or the above-mentioned kit to process the sample to be tested to obtain a treatment solution containing the Ni-polymethacrylate magnetic beads; step 2: treating the sample to be tested The enriched bacterial solution is subjected to DNA extraction treatment to obtain an extracted product; Step 3: Amplify the extracted product to obtain an amplified product; Step 4: Detect the amplified product so as to detect the sample to be tested whether it contains Staphylococcus aureus. As mentioned above, the use of the aforementioned adsorption magnetic beads or kits can specifically bind to Staphylococcus aureus, thereby realizing the capture and enrichment of Staphylococcus aureus, so there is no need to culture them, simplifying the detection steps and time, and improving the detection efficiency.

According to an embodiment of the present invention, step 1 further includes: combining the adsorption magnetic beads or pre-connected Ni-polymethacrylate magnetic beads and the Amidase 3-CBD protein with His-tag sequence with the sample to be tested Mixing and reaction were performed to obtain the treatment liquid. Thereby, the capture of Staphylococcus aureus in the sample to be tested can be achieved.

According to an embodiment of the present invention, the temperature of the reaction is 2-30°C. Thus, the capture efficiency of the adsorbed magnetic beads for Staphylococcus aureus can be improved.

According to an embodiment of the present invention, the pH of the reaction is 5-9. Thus, the capture efficiency of the adsorbed magnetic beads for Staphylococcus aureus can be improved.

According to an embodiment of the present invention, NaCl is added before the reaction, and the final concentration of the NaCl is 50-200 mM. Therefore, the binding effect of the Amidase 3-CBD protein on the adsorption magnetic beads and Staphylococcus aureus is better; and, the inventors have found through experiments that with the increase of the NaCl concentration, the binding effect of the protein and Staphylococcus aureus is constantly weakened .

According to an embodiment of the present invention, the DNA extraction treatment includes: boiling the treatment solution and endolysin with water. The inventor found through a large number of experiments that endolysin can specifically lyse the cell wall of Staphylococcus aureus, resulting in the release of intracellular substances of Staphylococcus aureus, and then the DNA in Staphylococcus aureus can be extracted by boiling, and can be extremely It greatly improves the extraction amount of Staphylococcus aureus DNA, and has the advantages of shortening extraction time, convenient operation, and large extraction amount.

According to an embodiment of the present invention, before the boiling, the endolysin and the treatment solution are reacted for 20-40 min. Thereby, the cell wall of Staphylococcus aureus can be fully lysed, and the amount of DNA extracted from Staphylococcus aureus can be increased.

According to an embodiment of the present invention, the boiling time is 5-20 min. Therefore, 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 the endolysin is 1-3 mg/ml. The inventor obtained the above-mentioned optimal ratio through a large number of experiments, and thus, the extraction effect of Staphylococcus aureus DNA was better.

It should be noted that the sample to be tested can be pre-treated before being treated with the above-mentioned adsorption magnetic beads or the above-mentioned kit. The pre-treatment includes but is not limited to dilution treatment and impurity removal treatment, and the specific method is not limited.

According to an embodiment of the present invention, the amplification treatment is performed using a recombinase polymerase amplification method. Therefore, the recombinase polymerase amplification (Recombinase Polymerase Amplification, RPA for short) method can work at a low temperature and constant temperature, does not require thermal denaturation of the template, does not need to use an instrument, and has the advantages of simple operation and the like. Specifically, the present invention does not strictly limit the specific steps and required reagents of the recombinase polymerase amplification method, which can be obtained by conventional means in the art.

According to an embodiment of the present invention, the detection includes: reacting the amplification product, Cas12a, crRNA and ssDNA-FQreporter; the reaction generates a detectable fluorescent signal, which is that the sample to be tested contains golden yellow grapes Indication of cocci; the reaction does not produce a detectable fluorescent signal, which is an indication that the sample to be tested does not contain Staphylococcus aureus. Among them, 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, and the target sequence fragment is cut first, and then the single strand is cut. ssDNA and produce a detectable fluorescent signal. Thus, at room temperature (30-40°C), the amplification product, Cas12a, crRNA and ssDNA-FQ reporter are mixed and reacted, and the DNA fragments in the amplification product can be quickly detected without the use of instruments, and the operation is simple .

According to an embodiment of the present invention, the crRNA has the nucleotide sequence shown in SEQ ID NO: 1 or a nucleotide sequence with more than 90% homology to the nucleotide sequence shown in SEQ ID NO. 1. The inventors screened a large number of crRNAs with different sequences and found that the above-mentioned crRNA activated the cas12a/crRNA complex to cleave the ssDNA-FQreporter with the strongest fluorescence value.

Additional aspects and advantages of the present invention will be set forth, in part, from the following description, and in part will be apparent from the following description, or may be learned by practice of the invention.

Description of drawings

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

1 is a schematic diagram of different domains and cloned fragments of 80α endolysin in Example 1 of the present invention;

Fig. 2 is the purification diagram of different domains of 80αendolysin in Example 2 of the present invention;

3 is a graph showing the analysis results of the binding situation of different proteins and Staphylococcus aureus detected by fluorescence microscopy and flow cytometry in Example 3 of the present invention;

Fig. 4 is the analysis result diagram of Western detection to different proteins and Staphylococcus aureus in Example 3 of the present invention;

Fig. 5 is the analysis result of the NaCl of different concentrations in the embodiment of the present invention 4 to EGFP-amidase 3-CBD protein stability;

Fig. 6 is the analysis result of EGFP-amidase 3-CBD protein stability at different temperatures in the embodiment of the present invention 4;

Fig. 7 is the analysis result of different pH to EGFP-amidase 3-CBD protein stability in Example 4 of the present invention;

Fig. 8 is the result diagram of capturing Staphylococcus aureus by Ni-polymethacrylate magnetic beads with Amidase 3-CBD protein in Example 5 of the present invention;

9 is a schematic diagram of Ni-polymethacrylate magnetic bead capture and recovery efficiency in Example 5 of the present invention;

Fig. 10 is the analysis result of endolysin+ boiled method and boiled method to extract the genomic DNA of Staphylococcus aureus in the embodiment of the present invention 6;

Fig. 11 is the agarose gel electrophoresis image after the NUC fragment of different bacteria is amplified by RPA method in Example 7 of the present invention;

Fig. 12 is the agarose gel electrophoresis image after the RPA method in the embodiment of the present invention 7 to different concentrations of Staphylococcus aureus genomic DNA amplification;

Figure 13 is an agarose gel electrophoresis image after the RPA method in Example 8 of the present invention has amplified the genomic DNA of Staphylococcus aureus in samples from different sources;

14 is an agarose gel electrophoresis image after purification of crRNA-1, crRNA-2 and crRNA-3 in Example 9 of the present invention;

Figure 15 is a schematic diagram of cas12a/crRNA cleavage detection and analysis of detection results in Example 9 of the present invention;

Figure 16 is the specificity analysis of Ni-polymethacrylate magnetic bead capture combined with cas12a/crRNA cleavage detection in Example 10 of the present invention;

Figure 17 is the detection result of Ni-polymethacrylate magnetic bead capture combined with cas12a/crRNA cleavage detection in Example 10 of the present invention.

Detailed ways

It should be noted that the terms "first" and "second" are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or implying the number of indicated technical features. Thus, a feature defined as "first" or "second" may expressly or implicitly include one or more of that feature. Further, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

The solution of the present invention will be explained below in conjunction with the embodiments. Those skilled in the art will understand that the following examples are only used to illustrate the present invention, and should not be construed as limiting the scope of the present invention. If no specific technique or condition is indicated in the examples, the technique or condition described in the literature in the field or the product specification is used. The reagents or instruments used without the manufacturer's indication are conventional products that can be obtained from the market.

Example 1: Bioinformatics analysis of 80α endolysin

The 80αEndolysin gene was found from the phage 80α genome (Genbank accession number DQ908929), the 80αEndolysin domain was analyzed with Interpro, and the molecular weight and isoelectric point of the protein were calculated using the pI/Mw program ExPASy.

The 80αEndolysin gene sequence was found from the phage 80α genome (Genbank accession number DQ908929), and the functional domain was searched with Interpro. The results showed that the 80αEndolysin gene consists of N-terminal CHAP catalytic domain, central amidase 3 catalytic domain and C-terminal The SH3b cell-binding domain consists of three domains, as shown in Figure 1. Therefore, the present invention subsequently selects two genes, Amidase 3-CBD and CBD, for cloning and expression.

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

The Amidase 3-CBD and CBD were cloned into the PET28a-EGFP vector, and the recombinant protein was obtained by purifying the E. coli gene expression system and affinity chromatography Ni-NTA column to obtain Amidase 3-CBD and CBD with EGFP, respectively. Named EGFP-Amidase 3-CBD, EGFP-CBD, and analyzed by SDS-PAGE.

As shown in Figure 2, the results show that the size of EGFP-Amidase 3-CBD protein is about 60kDa (that is, the purified product of lane 8 in Figure 2A), and the size of EGFP-CBD protein is about 40kDa (lane 7 in Figure 2B). the purified product), the protein sizes of both on SDS-PAGE gel matched the predicted 64 and 40.1 kDa bioinformatics (Fig. 2A and Fig. 2B), meanwhile, the negative control EGFP protein was detected on SDS-PAGE gel The size of the protein on (approximately 33 KDa) matched the bioinformatics prediction (33.2 KDa) (purified product of lane 8 in Figure 2C).

Example 3: EGFP-Amidase 3-CBD, EGFP-CBD binding activity analysis

Experimental method: Staphylococcus aureus was grown to mid-log phase under shaking culture at 37°C, then the culture was centrifuged at 12,000 rpm for 5 min, washed once with 50 mM Tris-HCl, and then centrifuged. The bacteria were resuspended with 50 mM Tris-HCl and the OD600 Adjust to 0.8. Take 200ul of bacterial liquid with OD600=0.8 in a centrifuge tube, add 100ul of EGFP-Amidase 3-CBD and EGFP-CBD protein with a concentration of 0.5mg/ml respectively, and incubate at room temperature for 20 minutes. Wash Staphylococcus aureus 3 times with 50 mM Tris-HCl by centrifugation (10000 rpm, 3 min) to remove unbound protein, and resuspend the washed pellet in 100 μL of 50 mM Tris-HCl to obtain a bacterial protein mixed suspension, Detection is performed by using fluorescence microscopy, flow cytometry, western.

Fluorescence microscopy: Add 10 μl of the final bacterial protein mixed suspension to a glass slide, cover with a coverslip, and pass through an epifluorescence microscope equipped with a U-RFL-T light source (1000x magnification) under brightfield and FITC The binding effect of the protein and Staphylococcus aureus was observed under the filter (excitation BP 470-490 nm; emission: LP 516 nm), and EGFP protein was prepared as a negative control at the same time. In order to evaluate the specificity and sensitivity of EGFP-Amidase 3-CBD, the bacteria listed as Staphylococcus aureus strains, Escherichia coli O157:H, Salmonella typHimurium, Bacillus cereus, Propionibacterium acnes, Listeria monocytogenes were incubated with the protein, respectively (The specific steps are the same as the above-mentioned experimental methods), according to the same procedure as above and observed under an epi-fluorescence microscope, the results are shown in Table 1.

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

Note: ^a +, with lytic activity; -, without lytic activity; ^b +, with cell wall binding activity; -, without cell wall binding activity.

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

Western detection: Add 5ul 5ⅹSDS loading buffer to 20ul final bacterial protein mixed suspension, boil at 100°C for 5 minutes, use SDS-PAGE precast gel for electrophoresis analysis, and then transfer the protein to PVDF membrane by membrane transfer technology. Western blot analysis was performed using anti-histidine antibody.

The results of fluorescence microscopy showed that the fusion proteins EGFP-Amidase 3-CBD and EGFP-CBD could bind to Staphylococcus aureus cells; the fluorescence intensity observation under the microscope showed that EGFP-Amidase 3-CBD had more Strong binding ability. However, no fluorescent cells were observed under the fluorescence microscope for the negative control EGFP, as shown in Figure 3A.

As shown in Figure 3B and Figure 3C, the detection results of flow cytometry showed that the peak value of EGFP-Amidase 3-CBD was significantly shifted to the right compared with the peak value of the positive control, and the average fluorescence intensity of EGFP-Amidase 3-CBD was higher than that of EGFP The average fluorescence intensity of -CBD is large, and there is a very significant difference between the two (P<0.0001), indicating that EGFP-Amidase 3-CBD has stronger binding ability than EGFP-CBD, and the negative control EGFP also showed average fluorescence in Figure 3C. Intensity value, the analysis is caused by not washing the unbound protein after protein binding to Staphylococcus aureus, which is a normal experimental error.

As shown in Figure 4, the gray value of EGFP-Amidase 3-CBD band is higher than that of EGFP-CBD, and it can also be seen from the comparison of non-specific bands that the non-specific band of EGFP-Amidase 3-CBD is higher than that of EGFP-CBD. EGFP-CBD is much lighter, indicating that EGFP-Amidase 3-CBD has stronger binding ability than EGFP-CBD.

Example 4: Study on the stability of EGFP-amidase 3-CBD protein

The stability of EGFP-amidase 3-CBD protein was investigated under the temperature range of 4-60°C, pH value of 3-12 and NaCl concentration of 0-1000mM. The protein treatment time was 30min under each condition, and 80α Endolysin was used. The C-terminal functional assay was used to determine the binding activity of amidase 3-CBD to Staphylococcus aureus.

Wherein, different pH conditions are realized by buffers with different pH: glycine-HCl pH=3, sodium acetate pH=5, Tris-HCl pH=7, glycine-NaOH pH=9, KCL-NaOH pH=12.

Among them, 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 analysis method of 80α Endolysin C-terminal.

As shown in Figure 5, the results showed that the binding ability of EGFP-amidase 3-CBD protein and Staphylococcus aureus was gradually weakened with the continuous increase of the concentration of NaCl after 100mM NaCl treatment. In the flow cytometry assay, the fluorescence peak and average fluorescence intensity of EGFP-amidase 3-CBD protein treated with 100 mM NaCl were the largest. When the NaCl concentration exceeded 200 mM, the binding capacity of EGFP-amidase 3-CBD decreased significantly.

As shown in Figure 6-7, the results of treating EGFP-amidase 3-CBD protein at different temperatures and pH show that when the temperature is 4 °C and pH=7, EGFP-amidase 3-CBD protein binds to Staphylococcus aureus the best effect.

Example 5: Investigation of the capture efficiency of Staphylococcus aureus by Ni-NTA magnetic beads with Amidase 3-CBD protein

Experimental method: Ni-polymethacrylate magnetic beads (Wuxi Biomag Biotechnology Co., Ltd.) have Ni-polymethacrylate groups on their surface, and Ni-polymethacrylate magnetic affinity ligands Ni ions can bind to the N-terminal 6×His-tag tag on the protein, and 100ul EGFP-amidase 3-CBD protein containing the His-tag tag (buffer formula: 50mMtris-HCl, 100mMNaCl, pH=7.4) Incubate with 100ul Ni-polymethacrylate magnetic beads combined in a mixer for 20min, in which EGFP-amidase3-CBD protein was added in excess compared to Ni-polymethacrylate magnetic beads; then separated by magnetic stand Ni-polymethacrylate magnetic beads, washed twice with T100. After washing twice with 50 mM Tris-HCl, the magnetic beads were finally resuspended in 100 ul of 50 mM Tris-HCl and stored at 4°C. The coating of Ni-polymethacrylate magnetic beads was observed by fluorescence microscope. Then the Ni-polymethacrylate magnetic beads coated with EGFP-amidase3-CBD protein were tested to capture the efficiency of Staphylococcus aureus, and the magnetic beads that captured Staphylococcus aureus were plated and counted. Staphylococcus (10 ⁴ -10 ¹ CFU/ml) was incubated with 100 ul magnetic bead suspension on a mixer for 40 min, the magnetic beads were separated by a magnetic stand, washed twice with 50 mM Tris-HCl, and finally the magnetic beads were resuspended in 1 ml The concentration of 50mM Tris-HCl, 1ml magnetic bead suspension was directly plated, after 24h incubation at 37°C, cells were counted by colony.

As shown in Figure 8, the inventors were pleasantly surprised to find that EGFP-amidase 3-CBD protein was very uniformly covered on the surface of Ni-polymethacrylate magnetic beads by fluorescence microscope observation, and after adding RFP-amidase 3-CBD It was observed that when the excitation light peaks were 488 nm and 532 nm, the observed green fluorescence, red fluorescence and the position of Staphylococcus aureus in bright field were one-to-one control. The above results show that the EGFP-amidase 3-CBD protein obtained by the present invention can easily achieve uniform coating on the surface of Ni-polymethacrylate magnetic beads.

As shown in Figure 9, Ni-polymethacrylate magnetic beads coated with EGFP-amidase 3-CBD protein exhibited a capture efficiency of 25% at 1×10 ⁴ CFU/ml of S. aureus bioburden ; As the bacterial load gradually decreases, the capture efficiency of magnetic beads is also increasing. At 1×10 ¹ CFU/ml, the capture efficiency of magnetic beads reaches 78%, which basically can achieve a good capture effect. .

Example 6: Investigation on the extraction of Staphylococcus aureus genome by different extraction methods

1. The steps of extracting Staphylococcus aureus genomic DNA by endolysin + boiling method are as follows:

The 80αEndolysin gene was analyzed and found from the phage 80α genome (Genbank accession number DQ908929), which was synthesized by BGI and cloned into the PMV vector. The 80αEndolysin gene was cloned into the PET28a vector by double enzyme digestion (BamHI/XhoI). The constructed PET28a-endolysin was transformed into BL21, 1 mM IPTG was added at OD600=0.6, and induced at 19°C for 20 h. 10000RPM, 5min to collect the induced bacteria, then suspend the bacteria in lysis buffer (50mM Tris-HCl, 500mMNaCl, 5mM imidazole), sonicate for 3min, centrifuge at 10000RPM, 30min to obtain soluble protein supernatant, and then purified by Ni-NTA to obtain The protein of interest, after which the protein was concentrated using Amicon Ultra-4 centrifugal filters Ultracel-30K, and the elution buffer (50 mM Tris-HCl, 500 mM imidazole, 100 mM NaCl) was replaced, and the protein was stored in 50 mM Tris-HCl. After purification, use sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) precast gel to analyze the protein, as shown in Figure 10A, the purified 80α endolysin protein is about 57KDa in SDS-PAGE analysis ( That is, the purified product of lane 9 in Figure 10A ), which matched the bioinformatically predicted size of the 80α endolysin protein (57.3 kDa).

Take 1mL of bacterial liquid with OD600=0.8 in a centrifuge tube, centrifuge at 10000rpm for 3min, discard the supernatant; add 100ul Tris-HCl (pH=7.4) buffer to suspend the bacteria; add 40ul 400ug/ml endolysin protein obtained above, The reaction was carried out for 30 min; the boiling water bath for 10 min, centrifuged at 10000 r for 3 min, and the supernatant was aspirated for qPCR.

Among them, the qPCR system is as follows:

genomic DNA Total volume 10ul Sybergreen mix 5ul Nuc(F): 5'-GGCATATGTATGGCAATTGTTTC-3' (SQE ID NO: 1) 0.3ul Nuc(R): 5'-CGTATTGCCCTTTCGAAACATT-3' (SQE ID NO: 2) 0.3ul H₂O 3.4ul

The cycle system is: 95°C, 15min; 95°C, 15s, 60°C, 20s, 30 cycles, 72°C, 45s.

2. The steps of extracting Staphylococcus aureus genomic DNA by boiling method are as follows:

Take 1 mL of bacterial liquid with OD600=0.8 in a centrifuge tube, centrifuge at 10000 rpm for 3 min, discard the supernatant; add 140 ul Tris-HCl (pH=7.4) buffer to suspend the bacteria; 100 ℃ water bath for 10 min, centrifuge 10000 r, 3 min, absorb The supernatant was subjected to qPCR.

Among them, the qPCR system is as follows:

genomic DNA Total volume 10ul Sybergreen mix 5ul Nuc(F): 5'-GGCATATGTATGGCAATTGTTTC-3' (SQE ID NO: 1) 0.3ul Nuc(R): 5'-CGTATTGCCCTTTCGAAACATT-3' (SQE ID NO: 2) 0.3ul H₂O 3.4ul

The cycle system is: 95°C, 15min; 95°C, 15s, 60°C, 20s, 30 cycles, 72°C, 45s.

As shown in Figure 10B, in the lysis experiment of endolysin protein (400ug/ml) on Staphylococcus aureus ATCC29213, 80α endolysin can reduce the absorbance at 600nm from 1.0 to 0.2 within 60 minutes at 400ug/ml, the results show that, Endolysin protein has good cleavage activity.

As shown in Fig. 10C and Fig. 10D, when the Ct Threshold in QPCR was set to 2000, the Ct values of endolysin + boiled method and boiled method were 10 and 15, respectively, which indicated that endolysin + boiled S. aureus genomic DNA The extraction amount is much higher than the boiling method. This is because the cell wall of Staphylococcus aureus is thicker, and the ordinary boiling method cannot effectively destroy the cell wall of Staphylococcus aureus, resulting in low efficiency of genomic DNA, while endolysin is encoded by phage Peptidoglycan hydrolase can specifically lyse the host bacterial cell wall, resulting in the release of intracellular substances. Therefore, the extraction yield of endolysin when combined with boiling method is higher than that of single boiling method, which provides a great advantage for subsequent rapid detection. very important role.

Example 7: RPA amplification of S. aureus genomic nuc fragments

RPA amplification of Staphylococcus aureus genome nuc fragment experimental method:

1. Pipette and mix 100ul Ni-polymethacrylate magnetic beads into an EP tube, wash twice with 500ul T100 for 2 min each time, discard the supernatant, and resuspend in 100ul T100.

2. Add 100ul of EGFP-amidase 3-CBD protein with a concentration of 1mg/ml and incubate for 20min. Every 2min, tilt the EP tube by 50° and gently rotate and shake by hand.

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

4. Centrifuge 3ml Staphylococcus aureus, wash once with 1ml Tris-HCl, adjust to OD600=0.6 with Tris-HCl, and then dilute to 1×10 ⁴ CFU/ml, 1×10 ³ CFU/ml, 1× 10 ² CFU/ml and 1×10 ¹ CFU/ml to obtain four dilutions of bacterial liquid.

5. Take 1ml of the above-mentioned four dilutions of bacterial solution in EGFP-amidase 3-CBD protein suspension, incubate for 20min, tilt the EP tube 50° every 2min, and gently rotate and shake by hand.

6. Put the incubated EGFP-amidase 3-CBD protein-Staphylococcus aureus suspension on a magnetic stand, discard the supernatant, add 500ul 50mM Tris-HCl to wash twice, 2min each time, and resuspend in 40ul 50mM Tris-HCl.

7. Add 10ul of 2mg/ml endolysin protein, incubate for 30min, then put it in a boiling water bath for 10min, and absorb the supernatant to obtain a crude extract.

8. The crude extract is subjected to RPA, and the specific steps are as follows:

The primers used in the RPA reaction were designed by Primer Premier 5.0, and the length of the primers was between 30nt and 35nt.

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&apos;-TCTCTACACCTTTTTTAGGATGCTTTGTTTCA-3&apos; (SQE ID NO: 6).

The reaction system of RPA is: 10uM upstream primer (RPA-1-F or RPA-2-F) and 10uM downstream primer (RPA-1-R or RPA-2-R), 50mM Tris-HCl (pH 7.5) , 100 mM potassium acetate, 14 mM magnesium acetate, 2 mM dithiothreitol (DTT), 5% polyethylene glycol, 200 uM dNTP, 3 mM ATP, 1 ul of 5000 units/ml Bsu DNA polymerase and 2 ul of amplification product. The above mixture was incubated in a conventional water bath at 37°C for RPA reaction, wherein the concentration of each substance in the above mixture was the final concentration, and RPA-1-F and RPA-1-R were amplified to obtain nuc fragment 1, RPA -2-F and RPA-2-R were amplified to obtain nuc fragment 2.

The experimental method of RPA amplification of E. coli genome nuc fragments is the same as that of RPA amplification of Staphylococcus aureus genome nuc fragments, the difference is only in the choice of bacteria.

The amplification products of the above Staphylococcus aureus and Escherichia coli were extracted with phenol/chloroform, respectively, and electrophoresed on a 1% agarose gel. The results are shown in FIG. 11 .

The results in Figure 11 show that clear bands can be seen in the S. aureus lanes, but no bands in the E. coli lanes, indicating that the nuc gene is a S. aureus-specific gene and can be isothermally amplified by RPA way to expand.

As shown in Figure 12, for the sensitivity of RPA, 10-fold serially diluted Staphylococcus aureus genomic DNA was used as a template for isothermal amplification of RPA, and agarose gel electrophoresis showed that the detection limit of RPA was 10 ² aM.

Example 8: Exploring whether the capture of Staphylococcus aureus by Ni-polymethacrylate magnetic beads with Amidase 3-CBD protein is affected by food matrix

Experimental method: The method in Example 7 was used to capture and extract Staphylococcus aureus genomic DNA from the samples, and 1×10 ⁴ CFU/ml Staphylococcus aureus was contaminated with Tris-HCl, milk, orange juice and Cheese, using Ni-polymethacrylate magnetic beads coated with EGFP-Amidase 3-CBD protein to bind and capture Staphylococcus aureus, using endolysin + boiling method to extract genomic DNA, followed by isothermal amplification of nuc fragments using RPA, And run on 1% agarose gel electrophoresis, as shown in Figure 13.

The results showed that contaminated Tris-HCl, milk, orange juice and cheese could amplify nuc fragments, and the brightness of the bands was almost the same. Therefore, the above test results show that the capture of Staphylococcus aureus by the magnetic beads of the present invention is not affected by the food matrix.

Example 9: cas12a/crRNA cleavage detection

Three different crRNAs (see Figure 15A) were selected to perform cas12a/crRNA cleavage detection on the nuc fragment 1 and nuc fragment 2 obtained in Example 7. The cas12a/crRNA cleavage detection method is as follows:

Mix the amplification products obtained in Example 7 with 500nM crRNA, 250nM Cas12a, 2.5uM ssDNA-FQ reporter (sequence is 5'-FAM-TTATT-BHQ-1-3'), 2μL NEB buffer 3.1, 3μL, Add ddH ₂ O to 20 μL. The reaction was performed at 37°C for 30 minutes on a qPCR machine, and fluorescence measurements were performed every 1 minute. For details, see Figures 14-15. Among them, crRNA-1 is used to recognize nuc fragment 1, crRNA-2 and crRNA-3 are used to recognize nuc fragment 2, and the crRNA is as follows:

As shown in Figure 15C, crRNA-1 activated cas12a/crRNA to cleave the ssDNA-FQ reporter with the strongest fluorescence value, which indicated that crRNA-1 was the best choice for the three crRNAs. As shown in Figure 15B, in the analysis of the different components of the cas12a/crRNA cleavage reaction, it can be seen that the fluorescence value can only be generated when all the solvents in the reaction system are added, which is indispensable.

Example 10: Magnetic bead capture combined with cas12a/crRNA cleavage to detect Staphylococcus aureus in milk

In order to rapidly detect Staphylococcus aureus in food and prevent the occurrence of foodborne diseases caused by Staphylococcus aureus, the upstream magnetic beads-EGFP-Amidase 3-CBD were used to capture Staphylococcus aureus, and combined with downstream For the detection of RPA and cas12a cleavage, under the irradiation of ultraviolet light, whether the food was contaminated was observed and evaluated with the naked eye, and the specificity and sensitivity of the method were investigated.

Test method: 500nM crRNA-1 (see Example 9 for details), 250nM Cas12a, 2.5uM ssDNA-FQreporter (sequence is 5'-FAM-TTATT-BHQ-1-3'), 2μL NEB buffer 3.1, 3μL The amplification products obtained in Example 7 were mixed, and ddH ₂ O was added to 20 μL. Reactions were performed at 37°C for 30 min on a qPCR machine with fluorescence measurements every 1 min.

Specificity investigation and results: Select 1×10 ⁴ CFU/ml Escherichia coli and Staphylococcus aureus to contaminate sterile milk, use the method in Example 7 to capture Staphylococcus aureus in the sample, and extract the Staphylococcus aureus genome DNA and RPA amplified the nuc fragment of S. aureus genome, and detected S. aureus specificity by cas12a/crRNA cleavage.

As shown in Figure 16, only Staphylococcus aureus activates cas12a, and cleaves ssDNA-FQ reporter to generate fluorescence value. The results show that the magnetic bead capture combined with cas12a/crRNA cleavage detection of the present invention can specifically detect Staphylococcus aureus.

Sensitivity investigation and results: Sterile milk was contaminated with 1×10 ³ CFU/ml, 1×10 ² CFU/ml and 1×10 ¹ CFU/ml Staphylococcus aureus respectively, and then 100ul EGFP-Amidase 3-CBD was added to coat the milk. The coated magnetic beads were used to extract the genomic DNA of Staphylococcus aureus by endolysin+water boiling method. After RPA reaction was carried out at 37°C for 30min, Cas12a was cut at 37°C for 30min. Under the irradiation of ultraviolet light, the fluorescence signal was checked by the naked eye; and the data were analyzed with GraphPad Prism 8.0 software and presented as the mean ± standard deviation, wherein the difference between the data means was analyzed by the t test, and when P<0.05, the indicates a significant difference.

As shown in Figure 17, Figures A, B, and C respectively show the detection of 1×10 ³ CFU/ml, 1×10 ² CFU/ml, 1×10 ¹ CFU/ml in milk by the method of magnetic bead capture combined with cas12a/crRNA cleavage. ml of Staphylococcus aureus, the curve in the figure is the fluorescence accumulation curve displayed by the cleavage reaction in the QPCR instrument set at 37°C for 30min, the test tube in the curve is the corresponding cut at 37°C for 30min and then irradiated with UV light The results show that the minimum detection limit of magnetic bead capture combined with cas12a/crRNA cleavage detection is 1×10 ¹ CFU/ml.

In the description of this specification, description with reference to the terms "one embodiment," "some embodiments," "example," "specific example," or "some examples", etc., mean specific features described in connection with the embodiment or example , structure, material or feature 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, those skilled in the art may combine and combine the different embodiments or examples described in this specification, as well as the features of the different embodiments or examples, without conflicting each other.

Although the embodiments of the present invention have been shown and described above, it should be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Embodiments are subject to variations, modifications, substitutions and variations.

SEQUENCE LISTING

<110> Chengdu University

<120> Adsorption magnetic beads, kits and uses thereof, and methods for detecting Staphylococcus aureus

<130> BI3212533

<160> 12

<170> PatentIn version 3.5

<210> 1

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> 1

<400> 1

ggcatatgta tggcaattgt ttc 23

<210> 2

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223> 2

<400> 2

cgtattgccc tttcgaaaca tt 22

<210> 3

<211> 32

<212> DNA

<213> Artificial Sequence

<220>

<223> 3

<400> 3

gcatcacaaa cagataacgg cgtaaataga ag 32

<210> 4

<211> 32

<212> DNA

<213> Artificial Sequence

<220>

<223> 4

<400> 4

acattaattt aaccgtatca ccatcaatcg ct 32

<210> 5

<211> 32

<212> DNA

<213> Artificial Sequence

<220>

<223> 5

<400> 5

catcacaaac aggtaacggc gtaaatagaa gt 32

<210> 6

<211> 32

<212> DNA

<213> Artificial Sequence

<220>

<223> 6

<400> 6

tctctacacc ttttttagga tgctttgttt ca 32

<210> 7

<211> 61

<212> RNA

<213> Artificial Sequence

<220>

<223> 7

<400> 7

uaauacgacu cacuauaggg uaauuucuac uaaguguaga uguugaaguu gcacuauaua 60

c 61

<210> 8

<211> 61

<212> DNA

<213> Artificial Sequence

<220>

<223> 8

<400> 8

taatacgact cactataggg taatttctac taagtgtaga tgttgaagtt gcactatata 60

c 61

<210> 9

<211> 63

<212> RNA

<213> Artificial Sequence

<220>

<223> 9

<400> 9

uaauacgacu cacuauaggg aauuucuacu guuguagaua uuaaagcgau ugauggugau 60

acg 63

<210> 10

<211> 63

<212> DNA

<213> Artificial Sequence

<220>

<223> 10

<400> 10

taatacgact cactataggg aatttctact gttgtagata ttaaagcgat tgatggtgat 60

acg 63

<210> 11

<211> 59

<212> RNA

<213> Artificial Sequence

<220>

<223> 11

<400> 11

uaauacgacu cacuauagga auuucuacua aguguagaua uuaaugucgc agguucuuu 59

<210> 12

<211> 59

<212> DNA

<213> Artificial Sequence

<220>

<223> 12

<400> 12

taatacgact cactatagga atttctacta agtgtagata ttaatgtcgc aggttcttt 59

Claims (9)

1. an adsorption magnetic bead, is characterized in that, described adsorption magnetic bead is selected from Ni-polymethacrylate magnetic bead, and described Ni-polymethacrylate magnetic bead passes through His-tag label sequence and Amidase 3- CBD protein linked; Optionally, a fluorescent group, preferably an EGFP group, is attached to the Amidase 3-CBD protein. 2. a test kit, is characterized in that, comprises: Ni-polymethacrylate magnetic beads; Amidase 3-CBD protein with His-tag sequence; Optionally, the Amidase 3-CBD protein is connected with a fluorescent group, preferably an EGFP group; Optionally, the Ni-polymethacrylate magnetic beads and the Amidase 3-CBD protein are provided in a manner of combining Ni and His-tag sequence. 3. Application of the adsorption magnetic beads according to claim 1 or the kit according to claim 2 in the detection of Staphylococcus aureus. 4. a method for detecting Staphylococcus aureus, characterized in that, comprising: Step 1: using the adsorption magnetic beads of claim 1 or the kit of claim 2 to process the sample to be tested, to obtain a treatment solution containing the Ni-polymethacrylate magnetic beads; Step 2: subjecting the treatment solution to DNA extraction to obtain an extraction product; Step 3: subjecting the extracted product to amplification processing to obtain an amplification product; Step 4: Detecting the amplification product, so as to detect whether the sample to be tested contains Staphylococcus aureus. 5. The method according to claim 4, wherein step 1 further comprises: mixing and reacting the adsorption magnetic beads or the pre-connected Ni-polymethacrylate magnetic beads and the Amidase 3-CBD protein with His-tag sequence with the sample to be tested to obtain the treatment solution; Optionally, the temperature of the reaction is 2-30°C; Optionally, the pH of the reaction is 5-9; Optionally, before the reaction, NaCl is added to the mixed system, and the final concentration of the NaCl is 50-200 mM. 6. The method according to claim 4, wherein the DNA extraction treatment comprises: boiling the treatment solution and endolysin; Optionally, before the boiling treatment, the endolysin and the treatment solution are reacted for 20-40min; Optionally, the time of the boiled 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 the endolysin is 1-3 mg/ml; Optionally, the endolysin is derived from a phage in the S. aureus. 7 . The method according to claim 4 , wherein the amplification treatment is performed by a recombinase polymerase amplification method. 8 . 8. The method of claim 4, wherein the detecting comprises: reacting the amplification product, Cas12a, crRNA and ssDNA-FQ reporter; The reaction produces a detectable fluorescent signal, which is an indication that the sample to be tested contains Staphylococcus aureus; The reaction does not produce a detectable fluorescent signal, which is an indication that the sample to be tested does not contain Staphylococcus aureus. 9. The method according to claim 8, wherein the crRNA has the nucleotide sequence shown in SEQ ID NO: 1 or is more than 90% identical to the nucleotide sequence shown in SEQ ID NO. 1 source nucleotide sequence.

Images