CN116794013A - Rapid detection method for staphylococcus aureus in ginkgo ketoester total mixed sample - Google Patents
Rapid detection method for staphylococcus aureus in ginkgo ketoester total mixed sample Download PDFInfo
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/65—Raman scattering
- G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons
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- Health & Medical Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
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- Immunology (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a preparation method of aptamer modified probe molecule loaded gold nano-star particles. The invention also provides an aptamer modified probe molecule loaded gold nanostar particle. The invention further provides application of the aptamer modified probe molecule loaded gold nanostar particles in staphylococcus aureus detection. The invention further provides a rapid detection method for staphylococcus aureus in the ginkgo ketoester total mixed sample. The rapid detection method for the staphylococcus aureus in the ginkgo ketoester total mixed sample provided by the invention realizes quantitative detection of the staphylococcus aureus, saves detection cost, improves detection speed, can be used for quantitative detection of pathogenic bacteria in medicines, has the advantages of high detection speed, wide detection range, high stability and sensitivity, and has wide application prospects and use values in the technical fields of medicine safety, food safety, environmental monitoring and the like.
Description
Technical Field
The invention belongs to the technical field of drug detection, and relates to a rapid detection method of staphylococcus aureus in a ginkgo ketoester total mixed sample, in particular to a rapid detection method of staphylococcus aureus in a ginkgo ketoester total mixed sample based on an aptamer modified probe molecule loaded gold nanostar surface enhanced Raman spectrum technology.
Background
Pathogenic microorganisms refer to microorganisms that can invade the human body, causing infection or even infectious disease. In nature, pathogenic microorganisms are countless. Common pathogenic microorganisms described in the chinese pharmacopoeia (2020 edition) mainly include escherichia coli, salmonella, staphylococcus aureus, pseudomonas aeruginosa, candida albicans, and the like. Drugs are important items for treating diseases, but microbial contamination often affects the safety and effectiveness of drugs. The traditional Chinese medicine is prepared from traditional Chinese medicinal materials, and most of the traditional Chinese medicinal materials are natural wild plants, animals or minerals, and have a large number of microorganisms, so that the traditional Chinese medicine is easy to grow and reproduce at proper temperature and humidity. The 2020 edition of Chinese pharmacopoeia adds a 'traditional Chinese medicine decoction piece microorganism limit inspection method' in the general rule part, and has important significance for quality management of traditional Chinese medicine microorganism sources. In addition to the raw materials, microbial contamination needs to be controlled in the process of preparing the traditional Chinese medicine. Therefore, there is an urgent need to develop a rapid, low-cost, high-sensitivity method capable of effectively detecting and identifying pathogenic bacteria, which has a very important meaning for preventing and controlling diseases caused by pathogenic bacteria and ensuring the safety of medicines.
The traditional method for detecting and identifying the pathogenic bacteria is based on the culture, and is simple and low in cost, but long in culture time, even some microorganisms are difficult to culture due to the severe growth conditions, so that the traditional method for detecting the microorganisms cannot meet the rapid detection requirement of medicine production. In recent years, detection technology has developed from a plate counting method with long time consumption to an enzyme-linked immunoassay (ELISA) technology with strong specificity and a Polymerase Chain Reaction (PCR) technology capable of detecting various pathogenic bacteria simultaneously, to a mass spectrometry method, a biosensor and other analysis technologies in modern common detection technologies, and the detection methods have developed rapidly.
The enzyme-linked immunosorbent assay (ELISA) is one of the most widely used pathogen detection methods, and is the most commonly used antibody/antigen trace detection technology. ELISA method is to detect pathogenic bacteria by utilizing specific binding action of antigen and antibody, and compared with traditional microorganism culture method, the ELISA method has shorter time and stronger specificity, but has higher cost, and only when the concentration of pathogen is high enough, the immunological detection can provide real-time information.
The PCR technology is the most commonly used molecular biology technology for detecting pathogenic bacteria, and by using the principle of semi-reserved replication and through circularly repeating the processes of high-temperature denaturation, low-temperature annealing and medium-temperature extension under the action of DNA polymerase, target DNA fragments can be amplified in vitro in a short time, the required time is short, the sensitivity is high, and various pathogenic bacteria can be detected. However, the technology cannot detect living bacteria, and false positive is easy to occur in the detection result.
The mass spectrometry has good application prospect in microorganism detection and identification. Matrix assisted laser desorption ionization time of flight mass spectrometry (matrix-assisted laser desorption ionization time of flight mass spectrometry, MALDI-TOF MS) is used to identify and type microorganisms by dissociative analysis of proteins. The technology is simple to operate and high in sensitivity, but the mass spectrogram database of the strain is still incomplete.
The biosensor is an instrument which is sensitive to biological substances and converts the concentration of the biological substances into electric signals for detection, and is an analysis tool or a system which is composed of immobilized biological sensitive materials as identification elements (including bioactive substances such as enzymes, antibodies, antigens, microorganisms, cells, tissues, nucleic acids and the like), proper physicochemical transducers (such as oxygen electrodes, photosensitive tubes, field effect tubes, piezoelectric crystals and the like) and signal amplifying devices. Depending on the classification of the transducer, common biosensors include colorimetric biosensors, fluorescent biosensors, electrochemical biosensors, piezoelectric biosensors, and surface-enhanced raman spectroscopy biosensors. Compared with the traditional pathogenic bacteria detection technology, the biosensor has the characteristics of high sensitivity, high specificity, quick response, multiplexing and the like. Therefore, it is worth further research and discussion.
Disclosure of Invention
In view of the shortcomings of the prior art, the invention aims to provide a method for rapidly detecting staphylococcus aureus in a ginkgo ketoester total mixed sample, which can realize rapid quantitative detection of staphylococcus aureus in the ginkgo ketoester total mixed sample based on a surface-enhanced Raman scattering spectrum technology, and has the advantages of high sensitivity, strong stability and rapid detection speed.
To achieve the above and other related objects, a first aspect of the present invention provides a method for preparing aptamer-modified probe molecule-loaded gold nanostar particles (aunss@dtnb-Apt), comprising: preparing gold seeds by adopting a sodium citrate reduction method, synthesizing gold nano-star particles by adopting a seed growth method by the gold seeds, loading probe molecules on the gold nano-star particles, and reacting the gold nano-star particles (AuNSs@DTNB) loaded with the probe molecules with an aptamer to enable the aptamer to be modified on the gold nano-star particles, thereby obtaining the gold nano-star particles (AuNSs@DTNB-Apt) loaded with the aptamer modified probe molecules.
The second aspect of the invention provides aptamer modified probe molecule-loaded gold nanostar particles prepared by the method.
The third aspect of the invention provides an application of the aptamer modified probe molecule-loaded gold nanostar particles in staphylococcus aureus detection.
The fourth aspect of the invention provides a rapid detection method for staphylococcus aureus in a ginkgo ketoester total mixed sample, which comprises the following steps:
1) Culturing a standard substance of staphylococcus aureus to obtain a reference substance solution;
2) And respectively incubating the ginkgo ketoester total mixed sample and a reference substance solution in the aptamer modified probe molecule loaded gold nanostar particles (AuNSs@DTNB-Apt) provided by the second aspect of the invention, respectively carrying out surface-enhanced Raman scattering (Surface enhancement of Raman scattering, SERS) test, and determining the content of staphylococcus aureus in the ginkgo ketoester total mixed sample by adopting a standard curve method.
As described above, the rapid detection method for staphylococcus aureus in ginkgo ketoester total mixed samples provided by the invention has the following beneficial effects:
(1) According to the rapid detection method for staphylococcus aureus in the ginkgo ketoester total mixed sample, provided by the invention, based on a surface-enhanced Raman scattering spectrum technology, raman probe molecules are modified on gold nanosatellites, the gold nanosatellites modified with the probe molecules are synthesized, the aptamer is modified on the gold nanosatellites, the competitive combination effect of the aptamer and the probe molecules on the gold nanosatellites is utilized to establish a SERS-based staphylococcus aureus quantitative detection standard curve, and the SERS-based staphylococcus aureus quantitative detection standard curve is applied to the detection of staphylococcus aureus in the ginkgo ketoester total mixed sample, so that the quantitative detection of staphylococcus aureus can be realized, the method is simple to operate, and the detection cost is saved.
(2) According to the rapid detection method for staphylococcus aureus in the ginkgo ketoester total mixed sample, provided by the invention, as the gold nano star particles are provided with a plurality of arms with sharp corners, the sharp corners can generate very high electric field amplification, and the strong hot spots can be realized, so that trace samples can be detected.
(3) The rapid detection method of staphylococcus aureus in the ginkgo ketoester total mixed sample provided by the invention can be used for quantitatively detecting pathogenic bacteria in medicines, has the advantages of short sample incubation time, high detection speed, wide detection range, high stability and sensitivity, and can be widely applied to the technical fields of medicine safety, food safety, environmental monitoring and the like.
Drawings
Fig. 1 shows a schematic diagram of a method for rapidly detecting staphylococcus aureus in a ginkgo ketoester total mixed sample.
Fig. 2 shows characterization graphs (a), (b) and (c) of aptamer-modified probe molecule-loaded gold nanosheets in example 1 of the present invention, wherein graph (a) is a transmission electron microscope graph of gold seeds and gold nanosheets, graph (b) is an ultraviolet spectrogram of gold seeds and gold nanosheets, and graph (c) is an ultraviolet spectrogram of gold nanosheets, gold nanosheets@dtnb and gold nanosheets@dtnb-Apt.
FIG. 3a shows a surface-enhanced Raman spectrum of different concentrations of Raman reporter DTNB in example 5 of the present invention.
Fig. 3b shows a raman intensity histogram of different concentrations of raman reporter DTNB in example 5 of the present invention.
FIG. 4a is a graph showing the surface enhanced Raman spectrum of the Raman reporter DTNB of example 6 of the present invention at different reaction times.
Fig. 4b shows a bar graph of raman intensities for different reaction times of the raman reporter DTNB in example 6 of the present invention.
FIG. 5a shows a surface enhanced Raman spectrum for the addition of different amounts of the aptamer in example 7 of the invention.
Fig. 5b shows a bar graph of raman intensities for different amounts of aptamer added to example 7 of the invention.
FIG. 6a shows a surface-enhanced Raman spectrum of the aptamer of example 8 of the present invention at different reaction times.
Fig. 6b shows raman intensity histograms for different reaction times of the aptamers in example 8 of the present invention.
FIG. 7a shows a surface-enhanced Raman spectrum for different bacterial additions in example 9 of the present invention.
FIG. 7b shows a bar graph of Raman intensity for different bacterial additions in example 9 of the present invention.
Fig. 8a shows surface enhanced raman spectra of different bacteria incubation times in example 10 of the present invention.
Fig. 8b shows raman intensity histograms for different bacterial incubation times in example 10 of the present invention.
Fig. 9 shows a bar graph of raman intensities of staphylococcus aureus at different concentrations in example 11 of the present invention.
FIG. 10 is a graph showing the surface-enhanced Raman spectrum of Staphylococcus aureus at various concentrations in example 12 of the present invention.
FIG. 11 shows a standard graph of Staphylococcus aureus at various concentrations in example 12 of the present invention.
Detailed Description
The first aspect of the invention provides a preparation method of aptamer modified probe molecule loaded gold nanostar particles (AuNSs@DTNB-Apt), comprising the following steps: preparing gold seeds by adopting a sodium citrate reduction method, synthesizing gold nano-star particles by adopting a seed growth method by the gold seeds, loading probe molecules on the gold nano-star particles, and reacting the gold nano-star particles (AuNSs@DTNB) loaded with the probe molecules with an aptamer to enable the aptamer to be modified on the gold nano-star particles, thereby obtaining the gold nano-star particles (AuNSs@DTNB-Apt) loaded with the aptamer modified probe molecules.
Preferably, the sodium citrate reduction method comprises: and (3) carrying out reduction reaction on the gold-containing compound and a reducing agent to obtain gold seeds.
More preferably, the gold-containing compound is selected from one of a tri-valence gold-containing acid or a tetra-valence gold-containing acid.
Further preferably, the gold-containing compound is a quaternary gold-containing acid.
Still further preferably, the gold-containing compound is tetrachloroauric acid (HAuCl) 4 )。
Most preferably, the tetrachloroauric acid is present in an aqueous solution, which is an aqueous solution of tetrachloroauric acid. The concentration of the tetrachloroauric acid in the tetrachloroauric acid aqueous solution is 0.5-2mM, such as 0.5-1mM, 1-2mM, and preferably 1mM (mmol/L).
More preferably, the ratio of the volume of the gold-containing compound to the volume of the reducing agent added is 40:5-6, such as 40:5-5.5, and 40:5.5-6 in particular.
More preferably, the gold-containing compound is heated to boiling before the reduction reaction is carried out.
More preferably, the reducing agent is trisodium citrate.
Further preferably, the reducing agent is an aqueous solution of trisodium citrate. The concentration of the trisodium citrate in the trisodium citrate aqueous solution is 0.5-1.5% by mass, specifically, 0.5-1.0%, 1.0-1.5% by mass, and preferably 1.0% by mass.
More preferably, the reduction reaction is stirred and boiled for 5-25min, specifically for 5-10min,10-20min, and 20-25min.
Further preferably, the stirring is magnetic stirring.
More preferably, the reduction reaction is followed by cooling to room temperature. The room temperature is 20-30 ℃.
More preferably, the gold seeds are in a reddish wine-colored liquid. The gold seeds are used for preparing gold nano-star particles.
Preferably, the seed growing method comprises: mixing the acid liquor, the gold seeds, the precipitant, the L-ascorbic acid and the gold-containing compound, and stirring to obtain the gold nano-star particles.
More preferably, the acid solution, the gold seeds, the precipitant and the L-ascorbic acid are added with the gold-containing compound in sequence and then mixed.
More preferably, the acid liquor, gold seeds, precipitant, L-ascorbic acid, gold-containing compound are added in a volume ratio of 0.1-0.3:1-3:1-3:0.5-1.5:150-250, preferably 0.2:2:2:1:200.
More preferably, the acid solution is an aqueous hydrochloric acid solution. The concentration of hydrochloric acid in the aqueous hydrochloric acid solution is 0.5-1.5M, preferably 1.0M.
More preferably, the precipitant is an aqueous silver nitrate solution. The concentration of silver nitrate in the silver nitrate aqueous solution is 1-3mM, preferably 2mM.
More preferably, the L-ascorbic acid is present in the form of an aqueous solution, which is an aqueous solution of L-ascorbic acid. The concentration of L-ascorbic acid in the aqueous L-ascorbic acid solution is 50-150mM, preferably 100mM.
More preferably, the gold-containing compound is tetrachloroauric acid.
Further preferably, the tetrachloroauric acid is present in an aqueous solution, which is an aqueous solution of tetrachloroauric acid. The concentration of the tetrachloroauric acid in the aqueous solution of the tetrachloroauric acid is 0.2-0.3mM, preferably 0.25mM.
More preferably, the rotational speed of the stirring is 500-1200rpm, preferably 600-1000rpm.
More preferably, the stirring time is between 10 and 40s, preferably 30s.
More preferably, the solution color of the stirred mixture changes from pale yellow to grey blue.
More preferably, the stirring is followed by a standing.
Further preferably, the time of the standing is 5 to 10 minutes. The rest is performed at room temperature.
More preferably, sodium Dodecyl Sulfate (SDS) is added to the gold nanosilicon particles.
Further preferably, the sodium dodecyl sulfate is present in the form of an aqueous solution, which is an aqueous solution of sodium dodecyl sulfate. The concentration of the sodium dodecyl sulfate in the sodium dodecyl sulfate aqueous solution is 0.5-1.5% by mass, and preferably 1.0% by mass.
Further preferably, the sodium dodecyl sulfate is added in an amount of 0.9 to 1.1mL, preferably 1mL.
More preferably, the gold nanostar particles are liquid.
Preferably, the loading of the probe molecules on the gold nanostar particles comprises: and mixing the gold nanoscin particles with probe molecules, standing for reaction, centrifuging, and taking a precipitate to obtain the gold nanoscin particles (AuNSs@DTNB) loaded with the probe molecules.
More preferably, the ratio of the gold nanostar particles to the added volume of probe molecules is 1:0.009-0.016, preferably 1:0.01.
More preferably, the probe molecule is 5,5' -dithiobis (2-nitrobenzoic acid) (DTNB).
Further preferably, the probe molecule is present as an ethanol solution, which is an ethanol solution of 5,5' -dithiobis (2-nitrobenzoic acid) (DTNB). The concentration of the 5,5' -dithiobis (2-nitrobenzoic acid) (DTNB) in the ethanol solution is 10 -6 -10 -1 M is preferably 10 -4 M。
More preferably, the temperature of the standing reaction is room temperature.
More preferably, the time of the standing reaction is 14 to 16min, preferably 15min.
More preferably, the rotational speed of the centrifugation is 3400-3500rpm, preferably 3500rpm.
More preferably, the centrifugation is for a period of time of 5-15min, preferably 15min.
The centrifugation is used for removing redundant probe molecules on the upper layer, and gold nano-star particles carrying the probe molecules and precipitated on the lower bottom are reserved.
More preferably, water is further added to the gold nano-star particles loaded with the probe molecules for dispersion, so as to obtain a gold nano-star particle (AuNSs@DTNB) solution loaded with the probe molecules. The water is added in an appropriate amount for further use.
Further preferably, the ratio of gold nanostar particles loaded with probe molecules to the volume of water added is 0.1-0.2:1, preferably 0.2:1.
Further preferably, the probe molecule-loaded gold nanostar particles (auss@dtnb) are liquid after being dispersed by water.
Preferably, the reaction of the probe molecule-loaded gold nanostar particles (aunss@dtnb) with an aptamer comprises: adding water to the gold nano-star particles (AuNSs@DTNB) loaded with probe molecules to disperse, mixing the obtained gold nano-star particle (AuNSs@DTNB) solution with an aptamer to react, centrifuging, and taking a precipitate to obtain the aptamer modified probe molecule loaded gold nano-star particles (AuNSs@DTNB-Apt).
More preferably, the ratio of the volume of gold nanostar particle (AuNSs@DTNB) solution to the volume of aptamer addition is 1:0.070-0.11, preferably 1:0.1.
More preferably, the nucleotide sequence of the aptamer is shown in SEQ ID NO.1:5'-SH-C6-TCCCTACGGCGCTAACCCCCCCAGTCCGTCCTCCCAGCCTCACACCGCCACCG TGCTACAAC-3' was purchased from Shanghai Co., ltd.
Further preferably, the aptamer is present as an aqueous solution, the concentration of the aptamer in the aqueous solution of the aptamer being 9-11 μm, preferably 10 μm.
More preferably, the temperature of the reaction is 36-38 ℃, preferably 37 ℃.
More preferably, the rotational speed of the reaction is 150-200rpm, preferably 180rpm.
More preferably, the reaction time is 110 to 150min, preferably 120min.
The aptamer is modified on gold nanostar particles through Au-S bonds.
More preferably, the rotational speed of the centrifugation is 3000-4000rpm, preferably 3500rpm.
More preferably, the centrifugation is for a period of time of 5-15min, preferably 15min.
The centrifugation was used to remove excess probe molecules and aptamers from the upper layer, leaving the AuNSs@DTNB-Apt precipitated at the bottom.
More preferably, water is further added to the aptamer modified probe molecule loaded gold nanostar particles (AuNSs@DTNB-Apt) for dispersion, so as to obtain an aptamer modified probe molecule loaded gold nanostar particle (AuNSs@DTNB-Apt) solution. For further use.
Further preferably, the ratio of the aptamer modified probe molecule loaded gold nanostar particles to the added volume of water is 0.1-0.2:1, preferably 0.1:1.
More preferably, the aptamer modified probe molecule loaded gold nanostar particles (AuNSs@DTNB-Apt) are liquid after being dispersed.
The water is purified water.
The second aspect of the invention provides aptamer modified probe molecule-loaded gold nanostar particles prepared by the method.
The third aspect of the invention provides an application of the aptamer modified probe molecule-loaded gold nanostar particles in staphylococcus aureus detection.
Preferably, the staphylococcus aureus is present in ginkgo ketoesters.
In a fourth aspect, the present invention provides a method for rapidly detecting staphylococcus aureus in a ginkgo ketoester total mixed sample, as shown in fig. 1, comprising the following steps:
1) Culturing a standard substance of staphylococcus aureus to obtain a reference substance solution;
2) And respectively incubating the ginkgo ketoester total mixed sample and a reference substance solution in the aptamer modified probe molecule loaded gold nanostar particles (AuNSs@DTNB-Apt) provided by the second aspect of the invention, respectively carrying out surface-enhanced Raman scattering (Surface enhancement of Raman scattering, SERS) test, and determining the content of staphylococcus aureus in the ginkgo ketoester total mixed sample by adopting a standard curve method.
Preferably, in step 1), the standard of staphylococcus aureus is stored at ultra-low temperature. In particular, the storage temperature is-80 ℃.
Preferably, in step 1), the culturing comprises: after resuscitating and culturing a staphylococcus aureus standard on an LB solid culture medium, selecting single bacterial colony, inoculating the single bacterial colony into a first LB liquid culture medium for first culture, transferring the single bacterial colony into a second LB liquid culture medium for second culture, centrifuging the obtained culture bacterial liquid, and taking precipitate for dispersion and gradient dilution to obtain a reference solution.
More preferably, the standard of staphylococcus aureus is ATCC 25923 staphylococcus aureus.
More preferably, the LB solid medium is a nutrient agar medium. The nutrient agar medium is a conventionally used nutrient agar medium and can be purchased from the market.
More preferably, the first LB liquid medium and the second LB liquid medium are nutrient broth media. The nutrient broth is a conventionally used nutrient broth and is commercially available.
More preferably, the resuscitating culture has a culture temperature of 36-37 ℃, preferably 37 ℃.
More preferably, the resuscitating culture is carried out overnight, for example, for a period of about 18 to 24 hours.
More preferably, the volume of the first LB liquid medium is 0.9-1.1mL, preferably 1mL.
More preferably, the culture temperature of the first culture is 35 to 38 ℃, particularly such as 35 to 37 ℃, 37 to 38 ℃, preferably 37 ℃.
More preferably, the rotational speed of the first cultivation is 160-200rpm, particularly such as 160-180rpm, 180-200rpm, preferably 180rpm.
More preferably, the first incubation is carried out overnight, for example, for 18-24 hours.
More preferably, the ratio of the transfer is 1:50-110, specifically such as 1:50-75, 1:75-110, preferably 1:100.
More preferably, the volume of the second LB liquid medium is 19-21mL, preferably 20mL.
More preferably, the culture temperature of the second culture is 36 to 38 ℃, particularly such as 36 to 37 ℃, 37 to 38 ℃, preferably 37 ℃.
More preferably, the second cultivation is performed for a cultivation time of 13 to 15 hours, preferably 14 hours. Culturing until the culture solution is turbid for later use.
More preferably, the rotational speed of the centrifugation is 10000-15000rpm, particularly 10000-13000rpm, 13000-15000rpm, 11000-15000rpm, preferably 12000-14000rpm.
More preferably, the centrifugation time is 1-11min, specifically 1-5min, 5-11min, preferably 5-10min.
More preferably, the temperature of the centrifugation is 3-5 ℃, particularly such as 3-4 ℃, 4-5 ℃, preferably 4 ℃.
More preferably, the dispersion is in a physiological saline solution.
Further preferably, the physiological saline (NaCl) solution is a sterile physiological saline solution. The sterile physiological saline solution is a conventionally used sterile physiological saline solution and is commercially available. Specifically, for example, a sterile physiological saline solution having a mass percentage concentration of 0.9% is used.
Most preferably, the volume of the sterile aqueous NaCl solution is 0.5-1.5mL, such as 0.5-1.0mL, 1.0-1.5mL, preferably 1.0mL.
More preferably, the gradient dilution is to dilute the control solution with a sterile physiological saline solution or a sterile PBS buffer. The sterile PBS buffer is a conventionally used sterile PBS buffer, and is commercially available. Specifically, for example, the buffer is a sterile PBS buffer of pH7.2-7.4 at 0.01M.
Further preferably, the dilution factor is 1-20, in particular 1-10, 1-20, preferably 10.
More preferably, the total number of colonies in the control solution is determined using plate counting. The plate counting method is a plate counting method of the total number of traditional colonies.
Further preferably, the total number of colonies in the control solution is at least 3 experiments.
Preferably, in the step 2), the ginkgo ketoester total mixed sample is a granule extracting solution subjected to the total mixing process in the production process of the ginkgo ketoester tablet.
Preferably, in step 2), the ratio of the total mixture of ginkgo biloba esters to the volume of the addition of the AuNSs@DTNB-Apt is between 1:4 and 6, preferably between 1:5.
Preferably, in step 2), the concentration of the reference solution is 10 1 -10 6 CFU/mL, particularly such as 10 1 -10 3 CFU/mL、10 3 -10 6 CFU/mL, preferably 10CFU/mL.
Preferably, in step 2), the ratio of the volume of the control solution to the volume of the addition of AuNSs@DTNB-Apt is 1-3:5, preferably 2:5.
Preferably, in step 2), the incubation temperature of the ginkgolide total mixed sample and/or control solution in AuNSs@DTNB-Apt is 36-37 ℃, preferably 37 ℃.
Preferably, in step 2), the incubation speed of the ginkgolide total mixed sample and/or control solution in AuNSs@DTNB-Apt is 160-200rpm, specifically such as 160-180rpm, 180-200rpm, preferably 180rpm.
Preferably, in step 2), the incubation time of the ginkgo ketoester total mixed sample in AuNSs@DTNB-Apt is not more than 14h, specifically such as 0-4h, 4-6h, 6-8h, 8-10h, 10-12h, 12-14h, preferably 6h.
Preferably, in step 2), the incubation time of the control solution in AuNSs@DTNB-Apt is 15-50min, specifically 15-30min, 30-50min, preferably 30min.
The AuNSs@DTNB-Apt exists in the form of an aqueous solution, and is an AuNSs@DTNB-Apt solution.
Preferably, in step 2), the surface enhanced raman scattering spectrum (Surface enhancement of Raman scattering, SERS) combines raman spectrum and nanotechnology, which means that the detected substance molecules are adsorbed on the nanoscale metal surface, so that the raman scattering signal of the substance molecules is enhancedTechniques. SERS spectrum can not only improve the Raman signal of the detected substance by 10 3 ～10 14 The fluorescent interference can be well avoided by the order of magnitude, and the fluorescent probe is particularly suitable for the analysis and detection of low-concentration samples.
Preferably, in step 2), the SERS test is performed in a portable raman spectrometer.
Preferably, in step 2), after the incubation, the solution is dropped onto the surface of a silicon wafer for performing SERS test.
Preferably, in step 2), the SERS test conditions are: the laser wavelength is 780-790nm, such as 780-785nm, 785-790nm, preferably 785nm; the laser power is 40-45%, preferably 40%; wave number range of 150-3300 cm -1 Preferably 200 to 3200cm -1 The method comprises the steps of carrying out a first treatment on the surface of the The exposure time is 2-3s, such as 1-2s, 2-3s, preferably 2s; the number of collection times is 2-5, specifically 2-3, 3-5, preferably 3.
Preferably, in step 2), the standard curve method means: and respectively transferring a series of reference substance solutions with different staphylococcus aureus concentrations, adopting a Raman spectrometer for sample injection analysis to obtain the linear relation between the logarithm of the staphylococcus aureus concentration in the reference substance solution and the Raman signal intensity, drawing a corresponding standard working curve, and calculating to obtain a regression equation of the standard working curve. And detecting the total mixed sample of the ginkgo ketoester subjected to incubation and the standard by adopting a Raman spectrometer, and substituting the obtained Raman signal intensity into a regression equation of the standard working curve to obtain the concentration of the standard staphylococcus aureus.
More preferably, the standard operating curve has a raman signal intensity on the ordinate and a staphylococcus aureus concentration on the abscissa.
The invention is further illustrated below in connection with specific examples, which are to be understood as being illustrative of the invention and not limiting the scope of the invention.
Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.
Example 1
1. Preparation of gold seeds
Preparation of gold seeds by sodium citrate reduction method, first 1mM HAuCl 4 40mL of the aqueous solution was heated to boiling with stirring, and 6mL of a 1% trisodium citrate solution was then added dropwise to the boiled HAuCl 4 In the water solution, magnetically stirring and continuously boiling for 15min to ensure complete reduction, wherein the obtained wine red liquid is golden seed No. 1; subsequently, gold seed # 1 was cooled to room temperature and used for gold nanostar preparation.
2. Synthetic gold nano-star particles
The seed growth method is adopted to synthesize the gold nano-star, namely 200 mu L of 1M HCl aqueous solution, 2mL of gold seeds 1#, 2mL of 2mM AgNO are processed at the rotation speed of 800rpm 3 The aqueous solution and 1mL of 100mM aqueous L-ascorbic acid solution were added sequentially to 200mL of 0.25mM HAuCl 4 In the aqueous solution, the mixture is stirred for 30s, the color of the solution is changed from light yellow to gray blue rapidly in a short time, the mixed solution is stood for 10min, and finally 1mL of 1% SDS aqueous solution is added into the prepared gold nano-star particles 1# for standby.
3. Gold nanostar particles for preparing supported probe molecules
Will 10 mu L10 -4 The DTNB ethanol solution of M was added to 1mL of gold nanostar particles 1# and allowed to stand at room temperature for 15min. Subsequently, the mixture was centrifuged at 3500rpm for 15 minutes, and the excess DTNB solution on the upper layer was removed to obtain AuNSs@DTNB1#. Finally, 1mL of purified water was added to 0.2mL AuNSs@DTNB1# and redispersed to obtain aunss@dtnb solution 1# for further use.
4. Preparation of aptamer modified probe molecule loaded gold nanostar particles
100 mu L of 10 mu M aptamer solution is added into 1mL of AuNSs@DTNB solution 1# and mixed at 37 ℃ and 180rpm for reaction for 2 hours, and due to the existence of sulfhydryl groups in the aptamer, the aptamer can be bonded on gold nano-star particles through Au-S bonds; and centrifuging at 3500rpm for 15min, removing excessive DTNB and aptamer solution at the upper layer, and obtaining precipitate at the bottom, namely AuNSs@DTNB-Apt1#. Finally, 1mL of purified water was added to 0.1mL of AuNSs@DTNB-Apt1# and redispersed to obtain AuNSs@DTNB-Apt1# for further use.
The gold nano seeds 1# and the gold nano star particles 1# are characterized by adopting a high-resolution transmission electron microscope, and the result is shown in figure 2. As shown by the transmission electron microscope of AuNPs and gold nanosatellites in the (a) of FIG. 2, the gold nanosatellite particle 1# has a remarkable star-shaped structure, which indicates that the gold nanosatellite particle 1# has been successfully prepared. Whereas, as shown by the transmission electron microscope of AuNPs and gold nanosatellites in fig. 2 (b), auNPs has a maximum absorption peak at 520nm or so, and exhibits a reddish-white color under visible light, and gold nanosatellites has a maximum absorption peak at 655nm, and exhibits a grayish blue color under visible light. In fig. 2, (c) is the ultraviolet spectrograms of gold nanostar, gold nanostar@dtnb and gold nanostar@dtnb-Apt, the maximum absorption peak is red shifted, and successful synthesis of gold nanostar@dtnb and gold nanostar@dtnb-Apt is seen.
Example 2
1. Preparation of gold seeds
Preparation of gold seeds by sodium citrate reduction method, first 2mM HAuCl 4 40mL of the aqueous solution was heated to boiling with stirring, and then 5mL of a 0.8% trisodium citrate solution was added dropwise to the boiled HAuCl 4 In the water solution, magnetically stirring and continuously boiling for 10min to ensure complete reduction, wherein the obtained wine red liquid is golden seed No. 2; subsequently, gold seed # 2 was cooled to room temperature and used for gold nanostar preparation.
2. Synthetic gold nano-star particles
The seed growth method is adopted to synthesize the gold nanostar, namely 100 mu L of 1.5M HCl aqueous solution, 1mL of gold seed No. 2 and 1mL of 1mM AgNO are processed at the rotation speed of 900rpm 3 The aqueous solution and 1mL of 150mM aqueous L-ascorbic acid solution were added sequentially to 150mL of 0.20mM HAuCl 4 In the aqueous solution, the mixture is stirred for 40s, the color of the solution is changed from light yellow to gray blue rapidly in a short time, the mixed solution is stood for 5min, and finally 1mL of 1.5% SDS aqueous solution is added into the prepared gold nano-star particles 2# for standby.
3. Gold nanostar particles for preparing supported probe molecules
Will 12 mu L10 -4 The DTNB ethanol solution of M was added to 1mL of gold nanosatellite particles 2# and allowed to stand at room temperature for 16min. Subsequently, the supernatant was centrifuged at 3400rpm for 10min, and the excess DTNB solution was removed to obtain AuNSs@DTNB2#. Finally, 1mL of purified water was added to 0.1mL AuNSs@DTNB2# and redispersed to obtain aunss@dtnb solution # 2 for further use.
4. Preparation of aptamer modified probe molecule loaded gold nanostar particles
75 mu L of 10 mu M aptamer solution is added into 1mL of AuNSs@DTNB solution 2# and the mixture is allowed to stand at 38 ℃ and 160rpm for reaction for 2.5 hours, and due to the existence of sulfhydryl groups in the aptamer, the aptamer can be bonded to gold nano-star particles through Au-S bonds; and then centrifuging for 10min at 3400rpm, removing excessive DTNB and aptamer solution at the upper layer, and obtaining the precipitate at the bottom, namely AuNSs@DTNB-Apt2#. Finally, 1mL of purified water was added to 0.2mL of AuNSs@DTNB-Apt2# and redispersed to obtain AuNSs@DTNB-Apt2# for further use.
Example 3
The standard strain of ATCC 25923 staphylococcus aureus preserved at-80 ℃ is taken out, resuscitated and cultured on nutrient agar medium serving as LB solid medium, cultured overnight at 37 ℃, single colony is selected and inoculated into 1mL of first LB liquid medium, and cultured at 37 ℃ and 180rpm for 21h and then cultured at 1:100 ratio was transferred to 20mL of nutrient broth as the second LB liquid medium and cultured at 37℃for 14h until the bacterial solution became turbid for later use. Finally, the culture broth was centrifuged at 13000rpm at 4 ℃ for 5min, the supernatant removed and redispersed in 1mL of sterile physiological saline solution to obtain control solution 1. The control solution 1 was serially diluted with a 10-fold concentration gradient, and the total number of colonies of the appropriate concentration gradient was determined by conventional plate counting to obtain the total number of colonies of the original bacterial liquid, and three parallel experiments were performed.
Example 4
After incubation of the ginkgolide total mixed sample with the reference solution 1 in example 3 in the aunss@dtnb-Apt solution 1# obtained in example 1, respectively, surface Enhanced Raman Scattering (SERS) testing was performed, and the content of staphylococcus aureus in the labeled ginkgolide total mixed sample was determined by a standard curve method.
Wherein 250. Mu.L of AuNSs@DTNB-Apt solution # 1 was injected into 12mL of a shaking tube, 50. Mu.L of the total mixture of labeled ginkgo biloba extract was added, and incubated at 37℃for 6h at 180 rpm. Simultaneously, 250. Mu.L of AuNSs@DTNB-Apt solution 1# was injected into 12mL of shake tubes, 100. Mu.L of control solution 1 was added, and incubated at 37℃for 30min at 180 rpm. And respectively taking 10 mu L of the total mixed sample of the control substance solution 1 after incubation and the ginkgo ketoester after labeling, dripping the total mixed sample onto the surface of a silicon wafer, and carrying out SERS (surface enhanced Raman spectroscopy) test in a Raman spectrometer.
SERS test conditions were: 785nm laser wavelength, 40% laser power and 200-3200 cm wave number -1 Exposure time was 2s and the number of acquisitions was 3.
The standard curve method is to respectively remove a series of reference substance solutions 1 with different staphylococcus aureus concentrations, sample injection analysis is carried out by adopting a Raman spectrometer, the linear relation between the logarithm of the staphylococcus aureus concentration and the Raman signal intensity in the reference substance solution 1 is obtained, a corresponding standard working curve is drawn, and a regression equation of the standard working curve is calculated. And detecting the total mixed sample of the ginkgo ketoester subjected to incubation and the standard adding by adopting a Raman spectrometer, and substituting the obtained Raman signal intensity into a regression equation of the standard working curve to obtain the concentration of the standard adding staphylococcus aureus.
Example 5
Preparation of AuNSs@DTNB, wherein different concentrations of DTNB were selected to be 10 respectively -1 M、10 -2 M、10 -3 M、10 -4 M、10 -5 M and 10 -6 M, other preparation conditions were the same as in example 1. The obtained AuNSs@DTNB-Apt was subjected to Raman spectroscopy under the conditions of example 4, and the results are shown in FIGS. 3a and 3 b. As shown in fig. 3a, 3b, add 10 -4 And the DTNB with M concentration has the strongest Raman signal of the detection result.
Example 6
AuNSs@DTNB was prepared with different reaction times of 15min, 30min, 45min, 60min, 90min, 120min for the selected DTNB, respectively, and other preparation conditions were the same as in example 1. The obtained AuNSs@DTNB-Apt was subjected to Raman spectroscopy under the conditions of example 4, and the results are shown in FIGS. 4a and 4 b. As shown in fig. 4a and 4b, the reaction time of DTNB was 15min, and the raman signal of the detection result was strongest.
Example 7
AuNSs@DTNB-Apt was prepared with different addition volumes of the selected aptamers of 25. Mu.L, 50. Mu.L, 75. Mu.L, 100. Mu.L, 150. Mu.L and 200. Mu.L, respectively, with the other preparation conditions being the same as in example 1. The obtained AuNSs@DTNB-Apt was subjected to Raman spectroscopy under the conditions of example 4, and the results are shown in FIGS. 5a and 5 b. As shown in FIGS. 5a and 5b, the addition volume of the aptamer was 100. Mu.L, and the Raman signal of the detection result was strongest.
Example 8
AuNSs@DTNB-Apt was prepared with different reaction times for the selected aptamers of 15min, 30min, 45min, 60min, 90min, 120min and 150min, respectively, with other preparation conditions as in example 1. The obtained AuNSs@DTNB-Apt was subjected to Raman spectroscopy under the conditions of example 4, and the results are shown in FIGS. 6a and 6 b. As shown in fig. 6a and 6b, the reaction time of the aptamer was 120min, and the raman signal of the detection result was strongest.
Example 9
Control solutions were prepared in which the different addition volumes of standard bacterial solutions for Staphylococcus aureus were 50. Mu.L, 100. Mu.L, 150. Mu.L, 200. Mu.L, 250. Mu.L and 300. Mu.L, respectively, under the same conditions as in example 3. The raman spectrum of the control solution obtained was collected under the conditions of example 4, and the results are shown in fig. 7a and 7 b. As shown in fig. 7a and 7b, the standard bacterial liquid of staphylococcus aureus was added in a volume of 50 μl, and the raman signal of the detection result was strongest.
Example 10
The control solution prepared in example 3 was used for 15min, 30min, 45min, 60min, 90min, 120min, 150min, and the other collection conditions were the same as in example 4, and the results are shown in fig. 8a and 8 b. As shown in fig. 8a and 8b, the raman signal intensity of the detection result is strongest when the incubation time is 30 min.
Example 11
In a total mixture containing 1g of ginkgo ketoesterStaphylococcus aureus was added to the LB medium to a final concentration of about 1 CFU/(1 g of ginkgo ketoester total mix), and cultured for 0h, 4h, 6h, 8h, 10h, 12h, and 14h, respectively. Subsequently, the solutions obtained at different incubation times were incubated with AuNSs@DTNB-Apt, respectively, and Raman signals were collected under the same conditions as in example 4, and the results are shown in FIG. 9. As shown in FIG. 9, 1336cm when the incubation time is 6h -1 The peak intensity at this point was significantly enhanced compared to 0h, thus 6h was the optimal incubation time for detection of staphylococcus aureus. Namely, staphylococcus aureus with the lowest detection concentration of 1 CFU/(1 g of ginkgo ketoester tablet total mixed particles) can be detected only through 6h of culture.
Example 12
The ginkgo ketoester total mixed sample (10) added with bacterial solutions with different concentrations 1 、10 2 、10 3 、10 4 、10 5 、10 6 CFU/mL) was incubated with gold nanostar @ DTNB-Apt solution, 10 μl of the above solution was dropped onto the silicon wafer surface for SERS test, and other collection conditions were the same as in example 4, and the results are shown in fig. 10.
And drawing a corresponding standard working curve according to the obtained concentration of a series of different bacterial liquids. As shown in fig. 11, in the standard curve of the logarithm of the raman signal intensity and the corresponding staphylococcus aureus concentration, the curve equation is y=2828×lgc+4996, r 2 = 0.9935, the linearity is good.
In conclusion, the rapid detection method for staphylococcus aureus in the ginkgo ketoester total mixed sample provided by the invention realizes quantitative detection of staphylococcus aureus, saves detection cost, improves detection speed, can be used for quantitative detection of pathogenic bacteria in medicines, has the advantages of high detection speed, wide detection range, high stability and sensitivity, and has wide application prospects and use values in the technical fields of medicine safety, food safety, environmental monitoring and the like. Therefore, the invention overcomes various defects in the prior art and has high industrial utilization value.
The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.
Claims (10)
1. A preparation method of aptamer modified probe molecule loaded gold nanostar particles comprises the following steps: preparing gold seeds by adopting a sodium citrate reduction method, synthesizing gold nano-star particles by adopting a seed growth method by the gold seeds, loading probe molecules on the gold nano-star particles, and then reacting the gold nano-star particles loaded with the probe molecules with an aptamer to enable the aptamer to be modified on the gold nano-star particles, so as to obtain the gold nano-star particles loaded with the aptamer modified probe molecules.
2. The method for preparing the aptamer modified probe molecule-supported gold nanostar particles according to claim 1, which is characterized by comprising any one or more of the following conditions:
a1 The sodium citrate reduction process comprises: carrying out reduction reaction on a gold-containing compound and a reducing agent to obtain gold seeds;
A2 The seed growth method comprises: mixing and stirring acid liquor, gold seeds, precipitant, L-ascorbic acid and a gold-containing compound to obtain gold nano star particles;
a3 The loading of probe molecules on gold nanostar particles comprises: mixing gold nanostar particles with probe molecules, standing for reaction, centrifuging, and collecting precipitate to obtain gold nanostar particles loaded with probe molecules;
a4 The reaction of the probe molecule-loaded gold nanostar particles with the aptamer comprises: and (3) adding water into the gold nano-star particles loaded with the probe molecules to disperse, mixing the obtained gold nano-star particle solution with the aptamer to react, centrifuging, and taking the precipitate to obtain the gold nano-star particles loaded with the aptamer modified probe molecules.
3. The method for preparing the aptamer modified probe molecule-supported gold nanostar particles according to claim 2, which is characterized by comprising any one or more of the following conditions:
a11 The gold-containing compound is selected from one of a tri-valence gold-containing acid or a tetra-valence gold-containing acid; preferably, the gold-containing compound is a quaternary gold-containing acid; more preferably, the gold-containing compound is tetrachloroauric acid;
a12 The ratio of the added volume of the gold-containing compound to the added volume of the reducing agent is 40:5-6;
A13 The reducing agent is trisodium citrate; preferably, the reducing agent is trisodium citrate aqueous solution, and the mass percentage concentration of trisodium citrate in the trisodium citrate aqueous solution is 0.5-1.5%;
a14 The reduction reaction is stirred and continuously boiled for 5 to 25 minutes;
a15 Cooling to room temperature after the reduction reaction;
a21 The volume ratio of the acid liquor, the gold seeds, the precipitant, the L-ascorbic acid and the gold-containing compound is 0.1-0.3:1-3:1-3:0.5-1.5:150-250;
a22 The acid liquor is hydrochloric acid aqueous solution; preferably, the concentration of silver nitrate in the silver nitrate aqueous solution is 1-3mM;
a23 The L-ascorbic acid is in the form of an aqueous solution, which is an aqueous solution of L-ascorbic acid; preferably, the concentration of L-ascorbic acid in the L-ascorbic acid aqueous solution is 50-150mM;
a24 The gold-containing compound is tetrachloroauric acid; preferably, the gold-containing compound exists in the form of an aqueous solution, and is an aqueous solution of tetrachloroauric acid, and the concentration of the tetrachloroauric acid in the aqueous solution of tetrachloroauric acid is 0.2-0.3mM;
a25 The stirring speed is 500-1200rpm;
a26 The stirring time is 10-40s;
a27 Standing after stirring; preferably, the standing time is 5-10min;
A28 Adding sodium dodecyl sulfate into the gold nano-star particles; preferably, the sodium dodecyl sulfate exists in the form of an aqueous solution, namely an aqueous solution of sodium dodecyl sulfate, wherein the mass percent concentration of the sodium dodecyl sulfate in the aqueous solution of sodium dodecyl sulfate is 0.5-1.5%, and the adding amount of the sodium dodecyl sulfate is 0.9-1.1mL;
a31 The ratio of the added volume of the gold nanostar particles to the added volume of the probe molecules is 1:0.009-0.016;
a32 The probe molecule is 5,5' -dithiobis (2-nitrobenzoic acid); preferably, the probe molecule is present as an ethanol solution, which is an ethanol solution of 5,5' -dithiobis (2-nitrobenzoic acid); more preferably, the concentration of DTNB in the ethanol solution of 5,5' -dithiobis (2-nitrobenzoic acid) is 10 -6 -10 -1 M；
A33 The temperature of the standing reaction is room temperature;
a34 The standing reaction time is 14-16min;
a35 The rotational speed of the centrifugation is 3400-3500rpm;
a36 The centrifugation time is 5-15min;
a37 Adding water into the gold nano-star particles loaded with the probe molecules for dispersion to obtain a gold nano-star particle solution loaded with the probe molecules; preferably, the ratio of the gold nanostar particles loaded with the probe molecules to the added volume of water is 0.1-0.2:1;
A41 The ratio of the added volume of the gold nano-star particle solution to the added volume of the aptamer is 1:0.070-0.11;
a42 The nucleotide sequence of the aptamer is shown as SEQ ID NO.1:5 '-SH-C6-TCCCTACGGCGCTAACCCCCCCAGTCCGTCCTCCCAGCCTCACACCGCCACCGTGCTACAAC-3'; preferably, the aptamer is in the form of an aqueous solution, and is an aqueous solution of the aptamer, wherein the concentration of the aptamer in the aqueous solution of the aptamer is 9-11 mu M;
a42 The temperature of the reaction is 36-38 ℃;
a43 The rotational speed of the reaction is 150-200rpm;
a44 The reaction time is 110-150min;
a45 The rotational speed of the centrifugation is 3000-4000rpm;
a46 The centrifugation time is 5-15min;
a47 Adding water into the gold nanoscin particles loaded by the aptamer modified probe molecules for dispersion to obtain a gold nanoscin particle solution loaded by the aptamer modified probe molecules; preferably, the ratio of the aptamer modified probe molecule loaded gold nanostar particles to the added volume of water is 0.1-0.2:1.
4. An aptamer modified probe molecule-loaded gold nanostar particle prepared by the method of any one of claims 1-3.
5. Use of an aptamer modified probe molecule-loaded gold nanostar particle according to claim 4 in detection of staphylococcus aureus.
6. A method for rapidly detecting staphylococcus aureus in a ginkgo ketoester total mixed sample comprises the following steps:
1) Culturing a standard substance of staphylococcus aureus to obtain a reference substance solution;
2) After the ginkgo ketoester total mixed sample and the reference substance solution are respectively incubated in the gold nanostar particles loaded by the aptamer modified probe molecules provided by the claim 4, respectively performing surface-enhanced Raman scattering spectrum test, and determining the content of staphylococcus aureus in the ginkgo ketoester total mixed sample by adopting a standard curve method.
7. The method for rapid detection of staphylococcus aureus in ginkgo ketoester total mixed samples according to claim 6, wherein in the step 1), the culturing comprises the following steps: after resuscitating and culturing a staphylococcus aureus standard on an LB solid culture medium, selecting single bacterial colony, inoculating the single bacterial colony into a first LB liquid culture medium for first culture, transferring the single bacterial colony into a second LB liquid culture medium for second culture, centrifuging the obtained culture bacterial liquid, and taking precipitate for dispersion and gradient dilution to obtain a reference solution.
8. The method for rapid detection of staphylococcus aureus in ginkgo ketoester total mixed samples according to claim 7, which is characterized by comprising any one or more of the following conditions:
B1 The standard of staphylococcus aureus is ATCC 25923 staphylococcus aureus;
b2 The LB solid culture medium is a nutrient agar culture medium;
b3 The first LB liquid medium and the second LB liquid medium are nutrient broth media;
b4 The culture temperature of the resuscitating culture is 36-37 ℃;
b5 The culture time of the resuscitating culture is overnight; preferably, the culture time of the resuscitating culture is 18-24 hours;
b6 The volume of the first LB liquid medium is 0.9-1.1mL;
b7 The culture temperature of the first culture is 35-38 ℃;
b8 The rotational speed of the first culture is 160-200rpm;
b9 The incubation time for the first incubation was overnight; preferably, the first culturing time is 18-24 hours; b10 The ratio of the transfer is 1:50-110;
b11 The volume of the second LB liquid medium is 19-21mL;
b12 The culture temperature of the second culture is 36-38 ℃;
b13 The culture time of the second culture is 13-15h;
b14 The rotational speed of the centrifugation is 10000-15000rpm;
b15 The centrifugation time is 1-11min;
b16 The temperature of the centrifugation is 3-5 ℃;
b17 The dispersion is in physiological saline solution; preferably, the volume of the sterile NaCl aqueous solution is 0.5-1.5mL; b18 The gradient dilution is to dilute the reference substance solution by adopting sterile physiological saline solution or sterile PBS buffer solution;
Preferably, the dilution factor is 1-20 times;
b19 The total number of colonies in the control solution was determined by plate counting.
9. The method for rapid detection of staphylococcus aureus in ginkgo ketoester total mixed samples according to claim 6, wherein in the step 2), any one or more of the following conditions are included:
c1 The ratio of the total mixed sample of ginkgo ketoester to the added volume of the gold nanostar particles loaded by the aptamer modified probe molecules is 1:4-6;
c2 The concentration of the reference solution is 10 1 -10 6 CFU/mL；
C3 The ratio of the added volume of the control solution to the added volume of the gold nanostar particles loaded by the aptamer modified probe molecules is 1-3:5; c4 The incubation temperature of the ginkgo ketoester total mixed sample and/or the reference substance solution in the gold nanostar particles loaded by the aptamer modified probe molecules is 36-37 ℃;
c5 The incubation rotating speed of the ginkgo ketoester total mixed sample and/or the reference substance solution in the gold nano-star particles loaded by the aptamer modified probe molecules is 160-200rpm;
c6 The incubation time of the ginkgo ketoester total mixed sample in the gold nano star particles loaded by the aptamer modified probe molecules is not more than 14h;
c7 The incubation time of the reference substance solution in the gold nano-star particles loaded by the aptamer modified probe molecules is 15-50min.
10. The method for rapidly detecting staphylococcus aureus in ginkgo ketoester total mixed samples according to claim 6, wherein in the step 2), the surface enhanced raman scattering spectrum test conditions are as follows: the laser wavelength is 780-790nm; the laser power is 40-45%; wave number range of 150-3300 cm -1 The method comprises the steps of carrying out a first treatment on the surface of the Exposure time is 2-3s; the collection times are 2-5 times.
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