Nanoprobe for detecting gram-positive bacteria as well as preparation method, detection method and application of nanoprobe

Abstract

The invention belongs to the technical field of gram-positive bacterium detection, and particularly relates to a nano-probe for detecting gram-positive bacteria and a preparation method, a detection method and application of the nano-probe for detecting the gram-positive bacteria, and the nano-probe for detecting the gram-positive bacteria comprises concanavalin A modified magnetic nanoparticles and vancomycin modified long afterglow nanoparticles, the magnetic nanoparticles comprise FeCl3, the long afterglow nanoparticles comprise ZnGa2O4: Cr, and the preparation method comprises a preparation method of the concanavalin A modified magnetic nanoparticles and a preparation method of the vancomycin modified long afterglow nanoparticles. The nano-probe provided by the invention has the functions of recognition, separation and enrichment, interference resistance and signal providing in the detection of gram-positive bacteria, and provides a new detection means for the recognition and detection of gram-positive bacteria.

Description

Technical Field

[0001] The invention belongs to the technical field of gram-positive bacteria detection, and specifically relates to a nanoprobe for detecting gram-positive bacteria and a preparation method, a detection method and an application thereof. Background Art

[0002] Gram-positive bacterial infections are clinically important and can easily cause a variety of diseases. It is worth noting that some Gram-positive bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), are resistant to multiple antibiotics, which makes treatment more complicated and their sensitivity to antibiotics is different from that of Gram-negative bacteria.

[0003] Staphylococcus aureus (abbreviated as Staphylococcus aureus) is a common foodborne pathogenic microorganism that is widely present in nature and the human body. It can invade the body and cause serious infection when the body's immunity is poor, and because of its drug resistance, the monitoring and treatment of Staphylococcus aureus in clinical practice is particularly important. In addition, Staphylococcus aureus can cause a variety of infectious diseases in the human body, including local suppurative infection, sepsis, food poisoning, etc., and is a zoonotic pathogen. Therefore, accurate detection of Staphylococcus aureus is of great significance for the prevention and control of diseases and food poisoning incidents caused by the bacteria. The detection method of Staphylococcus aureus is usually a traditional culture technique, which is time-consuming and cumbersome to operate. Other in vitro detection methods such as polymerase chain reaction (PCR) and liquid chromatography-tandem mass spectrometry have complex pre-treatment processes and certain requirements for personnel and instruments. Therefore, it is particularly important to develop simple, rapid and low-cost detection methods in primary hospitals. Summary of the invention

[0004] In order to solve the problems in the prior art, the present invention provides a nanoprobe for detecting Gram-positive bacteria and a preparation method, a detection method and an application thereof, so as to achieve the purpose of having the functions of identification, separation and enrichment, anti-interference and signal provision in the detection of Gram-positive bacteria, and providing a new detection means for identifying and detecting Gram-positive bacteria.

[0005] The present invention solves the technical problem by adopting the following technical solutions:

[0006] The present invention aims to provide a nanoprobe for detecting Gram-positive bacteria, comprising concanavalin A-modified magnetic nanoparticles and vancomycin-modified long-lasting-glow nanoparticles, wherein the magnetic nanoparticles comprise FeCl3 and the long-lasting-glow nanoparticles comprise ZnGa2O4:Cr.

[0007] Con A-modified magnetic nanoparticles (Con A-MNPs) are separation and enrichment nanoparticles with recognition function. The recognition molecule is Con A, which has the ability to recognize carbohydrate structures on bacterial cell walls. Magnetic nanoparticles (MNPs) provide separation and enrichment functions. Small molecule antibiotics show obvious selectivity for bacterial strains, and have excellent stability, good tolerance, cost-effectiveness and ready accessibility. Studies have found that vancomycin (Van) is a broad-spectrum glycopeptide antibiotic that can bind to D-Ala-D-Ala in the cell wall of most Gram-positive bacteria through five-point hydrogen bonds, showing significant selective targeting of Gram-positive (G + )The ability of bacteria.

[0008] Long afterglow nanoparticles (PLNPs) have a remarkable ability to capture excitation energy and dissipate this energy by slowly releasing captured carriers, producing sustained phosphorescence even in the absence of continuous excitation. This unique property of PLNPs enables them to effectively circumvent the confounding effects of autofluorescence in complex biological matrices. Therefore, the detection system based on PLNPs of the present invention produces a superior signal modulation ratio, which is conducive to obtaining more accurate and sensitive analysis results.

[0009] A method for preparing a nanoprobe for detecting gram-positive bacteria includes a method for preparing concanavalin A-modified magnetic nanoparticles and a method for preparing vancomycin-modified long-afterglow nanoparticles.

[0010] Furthermore, the preparation method of vancomycin modified long afterglow nanoparticles includes: dispersing long afterglow nanoparticles with a final concentration of 5 mg / mL in a sodium hydroxide solution with a concentration of 5 mM, stirring at room temperature for 24 hours, washing three times with deionized water, and vacuum drying to obtain hydroxylated long afterglow nanoparticles; dissolving the hydroxylated long afterglow nanoparticles in N,N-dimethylformamide, adding 3-aminopropyltriethoxysilane under electromagnetic stirring, placing in an 80°C oil bath, electromagnetically stirring for 24 hours, centrifuging, washing twice with N,N-dimethylformamide, washing once with anhydrous ethanol, and vacuum drying to obtain amino long afterglow nanoparticles; dissolving 5-100 μg of vancomycin in 2-(N-morpholino)ethanesulfonic acid (MES) buffer (pH 5.0) containing EDC (2-20 mg) and NHS (10-50 mg) 5.5) for 2 h, then, the amino-modified long afterglow nanoparticles with a concentration of 2 to 10 mg / mL were added to the above vancomycin mixture and stirred for another 6 h, the obtained long afterglow nanoparticles were centrifuged, and methoxypolyethylene glycol amine (MW750) was added to block at 37°C for 2 h to obtain vancomycin-modified long afterglow nanoparticles.

[0011] Furthermore, the final concentration of the hydroxylated long afterglow nanoparticles is 1.0 to 10.0 mg / mL, and the amount of 3-aminopropyltriethoxysilane is 1 to 50 μL per mL of the reaction solution.

[0012] Furthermore, the preparation method of long afterglow nanoparticles includes: mixing zinc nitrate, gallium nitrate and chromium nitrate in a molar ratio of 1:2:0.001-1 and stirring together, adjusting the total volume to 15 mL by adding deionized water, adjusting the pH to 8-10.5 by adding ammonia water to form a white precipitate, and then stirring for 0.5 hours, transferring the mixture to a polytetrafluoroethylene-lined autoclave (25 mL) and sealing it, first placing the autoclave in an oven at 220°C for 10 hours, and then naturally cooling to room temperature, and the white precipitate obtained after centrifugation is dispersed in 0.01M hydrochloric acid to form a transparent solution. In this step, possible zinc oxide impurities can be removed, and then mixed and washed with isopropanol, and finally vacuum dried to obtain long afterglow nanoparticles.

[0013] Further, the preparation method of concanavalin A modified magnetic nanoparticles includes: dispersing magnetic nanoparticles in anhydrous citric acid buffer, ultrasonicating for 10 minutes, stirring overnight at room temperature, and then washing three times with deionized water to obtain carboxylated magnetic nanoparticles; then dissolving the carboxylated magnetic nanoparticles in 2-(N-morpholino)ethanesulfonic acid buffer, adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide and sodium salt of N-hydroxysuccinimide sulfonate, reacting at room temperature for 2 hours to activate the carboxyl group, washing three times with PBS, mixing the activated magnetic nanoparticles (1 mg / mL) with 1-10 mg / mL concanavalin A in PBS containing 1 mM calcium chloride and 1 mM manganese chloride, stirring for 0.5-5 hours, and magnetically collecting to obtain concanavalin A modified magnetic nanoparticles. The magnetic nanoparticles are washed three times with a binding buffer (10 mM PBS with 1 mM CaCl2 and 1 mM MnCl2), and the obtained ConA-MNPs are stored at low temperature. After preparation, the concanavalin A-modified magnetic nanoparticles were added with 1% BSA and blocked at 37°C for 2 h.

[0014] Furthermore, the preparation method of magnetic nanoparticles includes: dissolving ferric chloride and trisodium citrate in ethylene glycol, then adding sodium acetate under stirring, stirring the mixture for 30 minutes, and then sealing it in a stainless steel autoclave lined with polytetrafluoroethylene, heating the autoclave at 200°C and maintaining it for 10 hours, and then cooling it to room temperature, washing the black product with ethanol and deionized water, and vacuum drying it to obtain magnetic nanoparticles.

[0015] A detection method for a nanoprobe for detecting Gram-positive bacteria, wherein Gram-positive bacteria are diluted to different concentrations, mixed with magnetic nanoparticles modified with concanavalin A and long-lasting nanoparticles modified with vancomycin, reacted for 1-10 minutes, subjected to magnetic separation for 1-10 minutes, and the phosphorescence intensity of a magnetic precipitation group and a magnetic supernatant group is detected by an ELISA instrument, and the intensity ratio is calculated to establish a standard curve of the intensity ratio and the concentration of the target analyte;

[0016] The clinical samples were mixed with concanavalin A-modified magnetic nanoparticles and vancomycin-modified long-afterglow nanoparticles. After reacting for 1-10 minutes, they were magnetically separated for 1-10 minutes. The phosphorescence intensity of the magnetic precipitation group and the magnetic supernatant group was detected by an enzyme marker. The intensity ratio was calculated and compared with the standard curve to obtain the concentration of the target detection object in the sample.

[0017] A nanoprobe for detecting Gram-positive bacteria, a preparation method of a nanoprobe for detecting Gram-positive bacteria, or a detection method of a nanoprobe for detecting Gram-positive bacteria and its application in detecting Staphylococcus aureus.

[0018] Compared with the prior art, the beneficial technical effects of the present invention are:

[0019] 1. The present invention modifies magnetic nanoparticles with a small molecule compound concanavalin A as an identification and separation enrichment particle, thereby simplifying the detection process and replacing the traditional centrifugation method; at the same time, vancomycin is modified on nanoparticles that emit red afterglow, which also serves as an identification molecule and a signal output molecule. The afterglow characteristics, anti-interference, and low background characteristics are utilized to improve the anti-interference ability of the detection, and the entire process is fast and simple.

[0020] 2. The present invention realizes the use of small molecule compound concanavalin A to modify magnetic nanoparticles and vancomycin to modify long afterglow nanoparticles, which significantly improves the detection speed, reduces the detection time and detection cost, greatly improves the convenience of detection and broadens its application scenarios.

[0021] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, it can be implemented in accordance with the contents of the specification. In order to make the above contents of the present invention and its objectives, features and advantages more obvious and easy to understand, the specific implementation methods of the present invention are listed below. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 TEM image of the magnetic nanoparticles in the present invention.

[0023] Figure 2 This is a TEM image of the long afterglow nanomaterial of the present invention.

[0024] Figure 3 This is a graph showing the potential changes before and after modification of the magnetic nanoparticles in the present invention.

[0025] Figure 4 This is a graph showing the potential change before and after the long afterglow nanomaterial is modified in the present invention. DETAILED DESCRIPTION

[0026] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary illustrations and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0027] In addition, unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0028] Example 1

[0029] The method for preparing magnetic nanoparticles comprises:

[0030] 1. Dissolve FeCl3 (0.2-1.0 g) and trisodium citrate (0.1-0.5 g) in ethylene glycol (5-10 mL).

[0031] 2. Then, NaAc (0.5-2.0 g) was added under stirring, and the mixture was vigorously stirred for 30 min, hydrothermally reacted at 200° C. for 10 h, and then cooled to room temperature.

[0032] 3. Wash the product with ethanol and deionized water several times.

[0033] The transmission electron microscopy images of the prepared magnetic nanoparticles are shown in Figure 1 , Figure 1 The prepared magnetic nanoparticles were prepared by adding 0.65 g of FeCl3, 0.2 g of trisodium citrate, 20 ml of ethylene glycol, and 1.2 g of NaAc.

[0034] Example 2

[0035] The preparation method of long afterglow nanoparticles comprises:

[0036] 1. Mix 1mmol Zn(NO3)2, 2mmol Ga(NO3)3, and 0.001-0.01mmol Cr(NO3)3 together and stir vigorously. Adjust the total volume to 15mL by adding deionized water.

[0037] 2. Rapidly add concentrated aqueous ammonia (28%) (about 1 mL) to adjust the pH to 8-10.5, and a white precipitate will form immediately. After stirring for another 0.5 h, the mixture is placed at 220°C for 10 h, and then cooled naturally until it reaches room temperature.

[0038] 3. The white precipitate obtained after centrifugation is dispersed in dilute hydrochloric acid to form a transparent solution. In this step, possible zinc oxide impurities can be removed. Then it is mixed and washed with excess isopropanol, and finally vacuum dried to obtain long afterglow nanoparticles.

[0039] The transmission electron microscope image of the obtained long afterglow nanoparticles is shown in Figure 2 , Figure 2 The long afterglow nanoparticles were prepared using 0.004 mmol of Cr(NO3)3 and the pH was adjusted to 9.2.

[0040] Example 3

[0041] The preparation method of concanavalin A modified magnetic nanoparticles comprises:

[0042] 1. Disperse 1-10 mg / mL magnetic nanomaterials into 0.2 M anhydrous citric acid buffer, ultrasonicate for 10 min, stir overnight at room temperature, and then wash three times with deionized water to obtain carboxylated magnetic nanoparticles.

[0043] 2. Dissolve 3-12 mg of carboxylated magnetic nanoparticles in 1-5 ml of 2-(N-morpholino)ethanesulfonic acid (MES) buffer, add 2-8 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 10-50 mg of N-hydroxysuccinimide sulfonic acid sodium salt, react at room temperature for 2 h to activate the carboxyl group, wash three times with PBS, mix the activated MNPs (1 mg / mL) with 0.1-10 mg / mL Con A in PBS containing 0.1-10 mM CaCl2 and 0.1-10 mM MnCl2, stir for 0.5-5 h, collect Con A-MNPs magnetically, wash three times with binding buffer (1 mM CaCl2 and 1 mM MnCl2 in 10 mM PBS), and store the obtained ConA-MNPs at low temperature.

[0044] 3. After the preparation of the Concanavalin A modified magnetic nanoprobe, BSA with a final concentration of 1% was added and blocked at 37°C for 2 hours to block the unbound sites and obtain the blocked Concanavalin A modified magnetic nanoprobe.

[0045] The potential changes before and after magnetic nanoparticle modification are shown in Figure 3 , Figure 3In the preparation of concanavalin A-modified magnetic nanoparticles, the magnetic nanomaterial was 10 mg / mL; in step 2, 4 mg of carboxylated magnetic nanoparticles were dissolved in 2 ml of 2-(N-morpholino)ethanesulfonic acid buffer, 6 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 20 mg of N-hydroxysuccinimide sulfonic acid sodium salt were added, and the activated MNPs (1 mg / mL) were mixed with 1 mg / mL Con A in PBS containing 1 mM CaCl2 and 1 mM MnCl2 and stirred for 1 h.

[0046] Example 4

[0047] The preparation method of vancomycin-modified long-lasting nanoparticles comprises:

[0048] 1. Disperse 30-180 mg of long afterglow nanomaterial in 20 mL of 5 mM sodium hydroxide solution, stir at room temperature for 24 h, wash three times with deionized water, and vacuum dry to obtain hydroxylated long afterglow nanoparticles.

[0049] 2. Disperse 30-80 mg of hydroxylated long afterglow nanoparticles in 20 mL of N,N-dimethylformamide, slowly add 10-1000 μL of 3-aminopropyltriethoxysilane under electromagnetic stirring, place in an 80°C oil bath, stir electromagnetically for 24 hours, then centrifuge and separate, wash twice with N,N-dimethylformamide and once with anhydrous ethanol, and vacuum dry to obtain amino-containing long afterglow nanoparticles.

[0050] 3. Activation of carboxyl groups: 5-100 μg of vancomycin was dissolved in 2-(N-morpholino)ethanesulfonic acid (MES) buffer (pH 5.5) containing 2.5-50 mg of EDC and 7.5 mg-150 mg of NHS for 2 h. Then, 2 mg / mL of amino-modified long-lasting nanoparticles were added to the above mixture and stirred for another 6 h. The obtained nanoparticles were centrifuged, washed to remove unattached carboxylated materials, and blocked with 1% mPEG-NH2 (MW750) at 37°C for 2 h to obtain vancomycin-modified long-lasting nanoparticle probes.

[0051] The potential changes before and after the modification of long afterglow nanoparticles are shown in Figure 4 , Figure 4 In the preparation of vancomycin modified long afterglow nanoparticles, 100 mg of long afterglow nanomaterials were dispersed in 20 mL of 5 mM sodium hydroxide solution; in step 2, 50 mg of hydroxylated long afterglow nanoparticles were dispersed in 20 mL of N,N-dimethylformamide, and 200 μL of 3-aminopropyltriethoxysilane was slowly added under electromagnetic stirring; in step 3, 20 μg of vancomycin was dissolved in 2-(N-morpholino)ethanesulfonic acid buffer containing 10 mg of EDC and 30 mg of NHS for 2 h.

[0052] Example 5

[0053] Taking Staphylococcus aureus (S. aureus) as an example, the detection methods for Gram-positive bacteria include:

[0054] 1. Take Staphylococcus aureus (S. aureus) bacterial suspension and dilute it to different concentrations. Take 50 μL of each as the experimental group and the control group as 50 μL H2O group. Add 50 μL of a mixture of 0.1-0.5 mg / mL ConA-MNP and 0.1-0.5 mg / mL PLNP-Van (25 μL of each of the two nanoparticles) to the two groups respectively. After 1-10 minutes of action, magnetic separation is performed for 1-10 minutes.

[0055] 2. The supernatant was aspirated and retained, and the precipitate was added with an equal volume of H2O as the supernatant. The phosphorescence intensity (time resolution) of the magnetic precipitation group and the magnetic supernatant group was detected by an ELISA instrument, the intensity ratio was calculated, and a standard curve of the intensity ratio and the concentration of the target analyte was established.

[0056] 3. Mix the clinical samples with magnetic nanoparticles modified with concanavalin A and long afterglow nanoparticles modified with vancomycin, react for 1-10 minutes, and then perform magnetic separation for 1-10 minutes. Use an ELISA instrument to detect the phosphorescence intensity (time resolution) of the magnetic precipitation group and the magnetic supernatant group, calculate the intensity ratio, and compare it with the standard curve to obtain the concentration of the target detection object in the sample.

[0057] The serial numbers of the above embodiments of the present invention are only for description and do not represent the advantages or disadvantages of the embodiments.

[0058] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation modes, which are merely illustrative rather than restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are within the protection of the present invention.

Claims

1. A nanoprobe for detecting Gram-positive bacteria, characterized in that: The invention comprises concanavalin A modified magnetic nanoparticles and vancomycin modified long afterglow nanoparticles, wherein the magnetic nanoparticles comprise FeCl3 and the long afterglow nanoparticles comprise ZnGa2O 4: Cr.

2. The method for preparing a nanoprobe for detecting Gram-positive bacteria according to claim 1, characterized in that: The invention comprises a preparation method of concanavalin A modified magnetic nanoparticles and a preparation method of vancomycin modified long afterglow nanoparticles.

3. A method for preparing a nanoprobe for detecting Gram-positive bacteria as claimed in claim 2, characterized in that: The preparation method of vancomycin modified long afterglow nanoparticles comprises: dispersing the long afterglow nanoparticles in a sodium hydroxide solution, stirring at room temperature, washing with deionized water three times, and vacuum drying to obtain hydroxylated long afterglow nanoparticles; dispersing the hydroxylated long afterglow nanoparticles in N,N-dimethylformamide, adding 3-aminopropyltriethoxysilane under stirring, placing in an 80°C oil bath and stirring for 24 hours, centrifuging, and washing with N,N-dimethylformamide and anhydrous ethanol to obtain amino-modified long afterglow nanoparticles; dissolving 5-100 μg of vancomycin in a 2-(N-morpholino)ethanesulfonic acid buffer containing EDC and NHS, then adding the amino-modified long afterglow nanoparticles to the above vancomycin mixture and stirring, resuspending the long afterglow by centrifugation, and adding methoxypolyethylene glycol amine group to seal for 2 hours to obtain vancomycin modified long afterglow nanoparticles.

4. A method for preparing a nanoprobe for detecting Gram-positive bacteria as claimed in claim 3, characterized in that: The final concentration of the hydroxylated long afterglow nanoparticles is 1.0-10.0 mg / mL, and the amount of 3-aminopropyltriethoxysilane is 1-50 μL per mL of the reaction solution.

5. The method for preparing a nanoprobe for detecting Gram-positive bacteria as claimed in claim 3, characterized in that: The preparation method of long afterglow nanoparticles includes: mixing zinc nitrate, gallium nitrate and chromium nitrate in a molar ratio of 1:2:0.001-1, adding ammonia water to adjust the pH to 8-10.5, continuing to stir for 0.5 hours, placing the mixture at 220°C for high pressure reaction for 10 hours, and then naturally cooling to room temperature. After centrifugation, the obtained precipitate is dispersed in isopropanol containing 0.01M hydrochloric acid, and washed 3 times to obtain long afterglow nanoparticles.

6. The method for preparing a nanoprobe for detecting Gram-positive bacteria as claimed in claim 2, characterized in that: The preparation method of concanavalin A modified magnetic nanoparticles comprises: dispersing the magnetic nanoparticles in anhydrous citric acid buffer, ultrasonicating, stirring at room temperature, and then washing three times with deionized water to obtain carboxyl-modified magnetic nanoparticles; then dissolving the carboxyl-modified magnetic nanoparticles in 2-(N-morpholino)ethanesulfonic acid buffer, adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide and sodium salt of N-hydroxysuccinimide sulfonate, reacting at room temperature to activate the carboxyl group, washing three times with PBS, mixing the activated magnetic nanoparticles with 0.1-10 mg / mL concanavalin A in PBS containing calcium chloride and manganese chloride, stirring for 0.5-5 hours, and collecting by magnetic separation to obtain the concanavalin A modified magnetic nanoparticles.

7. A method for preparing a nanoprobe for detecting Gram-positive bacteria as claimed in claim 6, characterized in that: The preparation method of magnetic nanoparticles includes: dissolving ferric chloride and trisodium citrate in ethylene glycol, then adding sodium acetate under stirring, stirring the mixture until dissolved, heating it at 200°C for 10 hours, then cooling it to room temperature, washing the black product with ethanol and deionized water, and vacuum drying it to obtain magnetic nanoparticles.

8. A method for detecting Gram-positive bacteria using a nanoprobe, characterized in that: Gram-positive bacteria were diluted to different concentrations, mixed with magnetic nanoparticles modified with concanavalin A and long-lasting nanoparticles modified with vancomycin, reacted for 1-10 minutes, and then magnetically separated for 1-10 minutes. The phosphorescence intensity of the magnetic precipitation group and the magnetic supernatant group was detected by an ELISA instrument, and the intensity ratio was calculated to establish a standard curve of the intensity ratio and the concentration of the target analyte. The clinical samples were mixed with concanavalin A-modified magnetic nanoparticles and vancomycin-modified long-afterglow nanoparticles. After reacting for 1-10 minutes, they were magnetically separated for 1-10 minutes. The phosphorescence intensity of the magnetic precipitation group and the magnetic supernatant group was detected by an enzyme marker. The intensity ratio was calculated and compared with the standard curve to obtain the concentration of the target detection object in the sample.

9. Use of the nanoprobe for detecting Gram-positive bacteria according to claim 1, the preparation method of the nanoprobe for detecting Gram-positive bacteria according to any one of claims 2 to 7, or the detection method of the nanoprobe for detecting Gram-positive bacteria according to claim 8 in detecting Staphylococcus aureus.

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