Method for rapidly and accurately detecting staphylococcus aureus in milk

Abstract

The invention discloses a method for rapidly and accurately detecting staphylococcus aureus in milk, and belongs to the technical field of microbiological detection.The method comprises the following steps that S1, a milk sample is pretreated; s2, staphylococcus aureus is enriched through immunocapture; s3, PMA treatment is carried out; step S4, extracting DNA (deoxyribonucleic acid); s5, real-time quantitative PCR detection: carrying out real-time quantitative PCR on the nuc gene, and quantifying the concentration of the active staphylococcus aureus through a standard curve; a milk sample to be detected is subjected to PMA treatment and DNA extraction to obtain a high-purity genome DNA solution, and real-time quantitative PCR detection is carried out. According to the scheme, the traditional culture step is replaced by immunocapture enrichment, and the bacterium enrichment time is shortened to 1 hour; by combining with a real-time quantitative polymerase chain reaction technology, electrophoresis detection after amplification is not needed, and the initial DNA template quantity can be directly calculated through fluorescence intensity change in the reaction process, so that accurate quantification is realized.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microorganism detection, and particularly relates to a rapid and accurate detection method for staphylococcus aureus in milk. BACKGROUND

[0002] Staphylococcus aureus is a common pathogenic bacterium and widely exists in the environment and food. In milk, staphylococcus aureus contamination may cause food poisoning, induce symptoms such as abdominal pain and diarrhea, and even produce drug-resistant strains such as methicillin-resistant staphylococcus aureus, which poses a serious threat to public health.

[0003] Traditional detection methods mainly include: 1. Culture method, but the culture method takes a long time (usually 24-48 hours), is not suitable for specific strains that cannot be cultured, and may underestimate the actual contamination risk. In addition, the culture method is complicated to operate and needs to be subjected to a series of identification steps, and is not suitable for large-scale screening.

[0004] 2. Staining color judgment method, which can only be qualitative or semi-quantitative, and cannot distinguish between live and dead bacteria.

[0005] 3. Molecular biology method, such as real-time quantitative polymerase chain reaction, which can quickly detect specific genes (such as nuc gene) of staphylococcus aureus, but cannot distinguish between live and dead bacteria, which may overestimate the infection risk, and the application in complex matrix (such as milk) has challenges, because the components of milk may interfere with the detection sensitivity, and there is a lack of standardized process for milk samples.

[0006] Therefore, it is necessary to develop a rapid and accurate detection method for staphylococcus aureus for milk, so as to realize high-sensitivity quantitative detection of active staphylococcus aureus. SUMMARY

[0007] In view of the deficiencies of the prior art, the application provides a rapid and accurate detection method for staphylococcus aureus in milk.

[0008] The application adopts the following technical means: The rapid and accurate detection method for staphylococcus aureus in milk comprises the following steps: Step S1, milk sample pretreatment: diluting and removing the upper lipid of the milk sample; Step S2, immune capture enrichment of staphylococcus aureus: co-incubating IgG cross-linked agarose microspheres with the milk sample, using the specific binding of the surface A protein of staphylococcus aureus and the Fc fragment of IgG to enrich the bacteria, and obtaining a microsphere-bacterium complex; Step S3, PMA treatment: adding PMA dye to the microsphere-bacterium complex, carrying out light incubation and light irradiation to inhibit the DNA amplification of dead bacteria, and obtaining a mixed solution containing the microsphere-bacterium complex; Step S4, DNA extraction: Extracting bacterial DNA from the microsphere-bacteria complex in the mixed solution to obtain a high-purity genomic DNA solution; Step S5, Real-time quantitative PCR detection: Real-time quantitative PCR is performed on the nuc gene to quantify the concentration of active Staphylococcus aureus by a standard curve; the high-purity genomic DNA solution obtained after PMA treatment and DNA extraction of the milk sample to be tested is subjected to real-time quantitative PCR detection.

[0009] Further, step S1 includes the following steps: Step S101, milk sample collection: Take 10 mL of the milk sample to be tested and store it at 4℃, and first centrifuge to remove the upper lipid; Step S102, dilution: Dilute the milk sample by 1:10 with sterile phosphate buffer to reduce the matrix effect; Step S103, filtration: The milk sample is preliminarily removed of large-particle impurities by a 0.45 μm filter membrane, and 20 mL of the milk sample is taken for standby.

[0010] Further, step S2 includes the following steps: Step S201, agarose microsphere preparation: 1, water phase preparation: Add 2% agarose by mass fraction to physiological saline containing 0.9% NaCl, microwave heat to clarify, and place in a 90℃ water bath to remove air bubbles to obtain a water phase; 2, oil phase preparation: Add 3% surfactant Span80 by volume fraction to liquid paraffin, heat to 70℃ and stir uniformly to obtain an oil phase; 3, emulsification: Mix the water phase and the oil phase at a volume ratio of 1:6, emulsify at 4000 rpm in a 60℃ water bath for 10 minutes; after emulsification, cool at 4℃ for 10 minutes to solidify the microspheres; 4, washing: Wash with deionized water, 20% ethanol and anhydrous ethanol by centrifugation, and finally filter through a 100 μm screen to collect agarose microspheres with a diameter of 50-100 μm, which are stored in 50% ethanol to obtain an agarose microsphere suspension with a concentration of about 10 6 ~ 10 7 microspheres / mL; Step S202, IgG cross-linking to obtain an IgG-agarose microsphere suspension: 1, take 2.5 mL of the agarose microsphere suspension, wash it twice with deionized water to remove ethanol residues; then add it to a 0.5 mol / L sodium carbonate solution containing 5% epichlorohydrin, and oscillate for 2 hours to activate the epoxy groups on the surface of the microspheres; 2, the sodium carbonate solution containing agarose microspheres, and the purified IgG solution is mixed, and oscillated for 24 hours, so that the IgG is cross-linked to the microspheres by a covalent bond, and the supernatant is removed by centrifugation to obtain IgG-agarose microspheres; 3, the IgG-agarose microspheres are added to a sodium carbonate solution containing 8.5% ethanolamine, non-specific adsorption is removed, and after stirring, an IgG-agarose microsphere suspension is obtained, which is reserved for later use; Step S203, capture incubation, to obtain a microsphere-bacteria complex: 1 mL of the IgG-agarose microsphere suspension is mixed with 20 mL of the pretreated milk sample, and incubated at 37°C on a shaking table at 100 rpm for 1 hour, so that the Staphylococcus aureus specifically binds to the IgG; After incubation, the microsphere-bacteria complex is collected by centrifugation at 1000 g for 5 minutes, and washed with PBS buffer for 3 times to remove the unbound components.

[0011] Further, step S3 includes the following steps: Step S301, PMA solution preparation: PMA powder is dissolved with sterile deionized water to prepare a PMA solution with a concentration of 1.5 mg / mL, which is stored at -20°C; Step S302, PMA solution addition: PMA solution is added to the microsphere-bacteria complex to obtain a mixed solution, and the concentration of the microsphere-bacteria complex is 1.5 μg / mL, which is stirred and mixed; Step S303, light-free incubation: the mixed solution is incubated at room temperature under light-free conditions for 5 minutes, so that PMA invades the damaged cell membrane of the dead bacteria; Step S304, light treatment: the mixed solution is placed on ice, and lighted for 20 minutes at a distance of 15 cm from a 500 W halogen lamp; light causes free PMA to photolyze into hydroxylamine, while PMA combined with the DNA of dead bacteria forms a covalent bond, inhibiting the amplification of dead bacteria DNA.

[0012] Further, step S4 includes the following steps: Step S401, pretreatment and resuspension of the microsphere-bacteria complex: DNA is extracted using a DNA extraction kit; 200 μL of pre-cooled PBS buffer is added to the mixed solution after PMA treatment at an ambient temperature of 4°C to obtain a sample to be extracted containing the microsphere-bacteria complex; the sample to be extracted is oscillated at medium speed for 30 seconds using a vortex oscillator to ensure that the microsphere-bacteria complex is completely resuspended, and the solution is observed to be uniformly turbid without visible clumps; Step S402, enzymatic lysis process: in the sample to be extracted, 20 μL of proteinase K solution is added, and the concentration of proteinase K is 200 μg / mL; the sample to be extracted is incubated in a water bath at 56°C for 30 minutes, and is gently inverted and mixed every 10 minutes during the incubation; it is observed that the solution changes from turbidity to clarity, indicating that the cell lysis is complete; Step S403, combined over column purification: after lysis, 200 μL of AL buffer is added to the sample to be extracted, and is shaken uniformly; 70°C incubation is performed for 10 minutes, and the sample to be extracted becomes clear and viscous; 200 μL of anhydrous ethanol is added, and is immediately shaken uniformly to prevent DNA precipitation; the entire solution of the sample to be extracted is transferred to a QIAamp centrifugal column, centrifugation conditions are 8000g and centrifugation for 1 minute, and DNA molecules are specifically combined on the surface of a silicon-based filter membrane of the QIAamp centrifugal column through hydrophobic interaction and ion exchange, and the solid after centrifugation is taken; Step S404, washing and elution; 1. The solid after centrifugation is washed; first washing: 500 μL of AW1 buffer is added, centrifugation conditions are 8000g and centrifugation for 1 minute, residual proteins, enzyme inhibitors and other polar impurities are removed, and a DNA-membrane complex is obtained; second washing: the DNA-membrane complex is added with 500 μL of AW2 buffer, centrifugation conditions are 14000g and centrifugation for 3 minutes, salt and organic solvent residues are completely removed, PCR inhibition is avoided, a high-purity DNA-membrane complex is obtained, higher centrifugal force ensures that ethanol is completely removed, and the subsequent reaction is prevented from being affected; then, emptying is performed, centrifugation conditions are 14000g and centrifugation for 1 minute, residual ethanol is completely removed, and a high-purity DNA-membrane complex is obtained; 2. The high-purity DNA-membrane complex is transferred from the centrifugal column to a collection tube with a volume of 2 mL; 50 μL of AE buffer preheated to 70°C is added to the collection tube, and the DNA solubility is enhanced; the collection tube is placed at room temperature for 5 minutes, the buffer is allowed to fully soak the membrane, and 8000g centrifugation is performed on the collection tube for 1 minute, the eluate is collected, and a high-purity genomic DNA solution is obtained.

[0013] Further, step S5 includes the following steps: Step S501, primer design: Target gene: nuc gene, which is a specific marker of Staphylococcus aureus; Primer sequence: upstream primer: 5'-GCGATTGATGGTGATACGGTT-3', downstream primer: 5'-AGCCAAGCCTTGACGAACTAAAGC-3'; Amplification fragment: 269 bp, which is verified to have species specificity through BLAST; Step S502, reaction system: total volume 20 μL, including 10 μL of SYBR Green fluorescent dye premix (2 times concentration), 0.5 μL of upstream primer, 0.5 μL of downstream primer, 5 μL of template DNA, 4 μL of nuclease-free water; The template DNA is extracted from PMA-treated DNA, and the concentration is in the range of 1-10 ng / μL; Step S503, real-time quantitative PCR treatment; Step S504, quantitative analysis: Standard curve preparation: standard curve is prepared with S. aureus standard, gradient dilution is 10-10 8 CFU / mL series concentration; with the logarithmic value of the standard concentration as the abscissa and the threshold cycle number Ct value as the ordinate, linear regression is performed; Sample concentration calculation: the Ct value of the sample to be tested is substituted into the standard curve equation to calculate the concentration value; the judgment standard is that the Ct value <35 is considered positive; the Ct value ≥35 or no detectable signal is considered negative.

[0014] The present application provides a rapid and accurate detection method of S. aureus in milk, which realizes the significant improvement of detection efficiency and accuracy by integrating immune capture enrichment and real-time quantitative polymerase chain reaction technology, and has the following core advantages: 1, time efficiency: shorten the detection time to 8 hours or less.

[0015] Limitations of traditional methods: traditional S. aureus detection mainly relies on culture method (such as plate counting method), which needs sample pretreatment, selective culture, colony separation, biochemical identification and other steps, and the whole process takes 24-48 hours. This method cannot meet the demand of rapid screening in dairy industry, especially in production process or epidemic emergency scene, which is easy to cause delay.

[0016] In this scheme, the traditional culture step is replaced by immune capture enrichment, and the bacterial enrichment time is shortened to 1 hour; combined with real-time quantitative polymerase chain reaction (real-time quantitative polymerase chain reaction) technology, without amplification electrophoresis detection, the initial DNA template amount can be calculated directly in the reaction process through fluorescence intensity change, realizing accurate quantification. DNA extraction and real-time quantitative polymerase chain reaction analysis only need 2-3 hours, and the whole process is controlled within 6-8 hours.

[0017] Practical significance: shortening the detection time can help dairy enterprises to realize the detection of raw materials or finished products on the same day, quickly judge the source of pollution, reduce the risk of product recall, especially suitable for raw milk procurement, production line monitoring and other scenes with high time efficiency requirements.

[0018] 2, Live bacteria specificity: distinguish live bacteria from dead bacteria, detection limit up to 10 CFU / mL.

[0019] The conventional quantitative polymerase chain reaction technology cannot distinguish the source of DNA (live bacteria or dead bacteria), and false positives may occur due to residual DNA from dead bacteria, overestimating the risk of contamination. For example, DNA from dead bacteria can still be detected in milk after pasteurization, misleading the quality judgment.

[0020] In this scheme, PMA is used to selectively inhibit dead bacteria: PMA (propidium monoazide) is a photoactivated dye that can penetrate the damaged cell membrane of dead bacteria and covalently bind to DNA under light, making the DNA of dead bacteria unable to be recognized and amplified in subsequent amplification reactions; the cell membrane of live bacteria is intact, PMA cannot enter, so only live bacteria DNA is detected.

[0021] In this scheme, the parameters are optimized to ensure sensitivity: the PMA concentration is preferably 1.5-2.3 μg / mL, the light incubation time is 5 minutes, and the distance from the 500W halogen lamp is 15 cm for 20 minutes of light. Under this condition, the dead bacteria inhibition rate is >99%, and the live bacteria signal retention rate is >95%.

[0022] Real-time quantitative polymerase chain reaction, primers are designed for nuc gene, detection limit up to 0.005 fg / μL of DNA, equivalent to 10 CFU / mL of bacteria solution concentration.

[0023] In a mixed sample with a dead bacteria ratio as high as 90%, this scheme can still accurately quantify live bacteria with an error rate of <5%.

[0024] Application value: It can accurately evaluate the real-time biological risk of milk and avoid misjudgment caused by dead bacteria interference, and is especially suitable for evaluating the effect of sterilization process or tracking the transmission route of active pathogenic bacteria.

[0025] 3, Improve specificity and accuracy in complex milk matrix.

[0026] Challenge of milk matrix: Milk is rich in fat, protein and miscellaneous bacteria, which may non-specifically bind to detection reagents or inhibit real-time quantitative polymerase chain reaction, leading to false negatives / false positives. For example, milk fat may encapsulate bacteria, reducing capture efficiency.

[0027] Anti-interference design of this scheme: immunocapture is used to improve specificity, IgG-agarose microspheres target Staphylococcus aureus A protein, and have no cross-reaction with common miscellaneous bacteria (such as Escherichia coli and Streptococcus). After optimization of microsphere surface modification, non-specific adsorption can be reduced, and the signal-to-noise ratio can be improved to 20:1.

[0028] Counteracting matrix effect of real-time quantitative polymerase chain reaction: Before light irradiation, the dye can be non-specifically adsorbed on sample impurities (such as proteins), generating background fluorescence. The PMA light irradiation step can photolyze the excess dye, reducing background noise.

[0029] The pretreatment step (dilution + PBS washing) reduces fat interference, and the recovery rate is increased to 85%-90%.

[0030] Technical advantages: This scheme still maintains high robustness in complex matrix, is suitable for different types of dairy products such as whole milk and skim milk, and avoids decision-making errors caused by false results.

[0031] 4, simple operation, suitable for on-site rapid screening.

[0032] Operation bottleneck of traditional method: culture method, need sterile operation, and need professional equipment such as constant temperature incubator.

[0033] This scheme, immunocapture and PMA treatment can be completed in the same centrifugal tube without sample transfer; the reagent of real-time quantitative polymerase chain reaction is premixed into freeze-dried powder form, which can be transported at room temperature, and can be used after activation with water. The whole process only needs a centrifuge, a portable real-time quantitative PCR instrument and a light irradiation device, and the equipment demand is simple. It can be combined with a portable nucleic acid extractor to realize on-site DNA extraction, which is suitable for remote scenes such as pastures and processing plants, and helps the grass-roots units to realize real-time monitoring of Staphylococcus aureus. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 The flowchart of the present application is shown. DETAILED DESCRIPTION

[0035] The technical solutions in the embodiments of the present application will be described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor belong to the scope of protection of the present application.

[0036] Figure 1 The flowchart of the present application is shown. Figure 1 As shown, the method for rapid and accurate detection of Staphylococcus aureus in milk includes the following steps: Step S1, pretreatment of milk sample: dilute the milk sample and remove the upper lipid.

[0037] Step S101, milk sample collection: take 10 mL of the milk sample to be tested, store at 4℃ and process as soon as possible. If the milk sample contains high fat, centrifuge (for example, 3000g) for 10 minutes to remove the upper lipid.

[0038] Step S102, dilution: dilute the milk sample with sterile phosphate buffer solution (PBS, pH 7.4) at a ratio of 1:10 to reduce the matrix effect.

[0039] Step S103, filtration: the milk sample is preliminarily removed from large particle impurities by a 0.45 μm filter membrane. Take 20 mL of the milk sample for standby.

[0040] Principle: pretreatment can reduce the interference of proteins and fats in milk on the capture efficiency, and improve the detection sensitivity. Sterile phosphate buffer solution maintains the osmotic pressure and avoids bacterial damage.

[0041] Step S2, immunocapture enrichment of Staphylococcus aureus: IgG cross-linked agarose microspheres are used to co-incubate with the milk sample, and the specific binding of the surface A protein of Staphylococcus aureus to the Fc fragment of IgG is used to enrich the bacteria.

[0042] The IgG cross-linking conditions are an epichlorohydrin concentration of 5% and a pH of 9.5.

[0043] Step S201, preparation of agarose microspheres: Agarose microspheres are spherical particle substrates prepared from agarose as the main raw material. Agarose is a natural linear polysaccharide polymer extracted from seaweed red algal plants (such as gelidium, etc.).

[0044] 1. Preparation of aqueous phase: add 2% agarose by mass fraction to physiological saline containing 0.9% NaCl, microwave heat to clarify, and place in a 90°C water bath to remove air bubbles to obtain the aqueous phase. The agarose concentration is preferably 2%-4% to ensure the mechanical strength of the microspheres.

[0045] 2. Preparation of oil phase: add 3% surfactant Span80 by volume fraction to liquid paraffin, heat to 70°C and stir uniformly to obtain the oil phase.

[0046] 3. Emulsification: mix the aqueous phase and the oil phase at a volume ratio of 1:6 in a 60°C water bath at 4000 rpm for 10 minutes; after emulsification, cool at 4°C for 10 minutes to solidify the microspheres.

[0047] 4. Washing: centrifugal washing with deionized water, 20% ethanol and anhydrous ethanol (centrifugal conditions: 1900g, 5 minutes / time), and finally filtered through a 100 μm screen to collect agarose microspheres with a diameter of 50-100 μm, stored in 50% ethanol to obtain an agarose microsphere suspension with a concentration of about 10 6 ~10 7 microspheres / mL.

[0048] Step S202, IgG cross-linking, to obtain an IgG-agarose microsphere suspension: 1, Take 2.5 mL of agarose microspheres suspension, wash twice with deionized water (centrifugal conditions: 2500g, 5 minutes / time) to remove ethanol residues; then add to 0.5 mol / L sodium carbonate solution containing 5% epichlorohydrin, shake for 2 hours to activate the surface epoxy group of the microspheres.

[0049] 2, Mix the sodium carbonate solution containing agarose microspheres with the purified IgG solution (concentration 3.2 mg / mL, solvent PBS buffer), shake for 24 hours to covalently cross-link IgG to the microspheres, and centrifuge to remove the supernatant to obtain IgG-agarose microspheres. IgG is the abbreviation of Immunoglobulin G.

[0050] PBS buffer is phosphate buffered saline solution.

[0051] 3, Add IgG-agarose microspheres to sodium carbonate solution containing 8.5% ethanolamine to remove non-specific adsorption, stir to obtain IgG-agarose microspheres suspension, and store for later use.

[0052] This step can ensure that the IgG binding efficiency is more than 90% and the microspheres have good stability.

[0053] Step S203, capture incubation, to obtain microsphere-bacteria complex: Take 1 mL of IgG-agarose microspheres suspension (about 10 6 Mix with 20 mL of pretreated milk sample, incubate at 37°C on a shaker at 100 rpm for 1 hour to allow specific binding of Staphylococcus aureus to IgG.

[0054] After incubation, centrifuge at 1000g for 5 minutes to collect the microsphere-bacteria complex, wash with PBS buffer for 3 times to remove the unbound components.

[0055] Principle: Staphylococcus aureus surface protein A binds to the Fc fragment of IgG with high affinity, achieving specific enrichment, and the enrichment efficiency can reach 80%-90%, significantly improving the subsequent detection sensitivity.

[0056] Step S3, PMA treatment: add PMA dye, perform light incubation and light irradiation to inhibit dead bacteria DNA amplification.

[0057] Step S301, PMA solution preparation: dissolve PMA powder with sterilized deionized water to prepare PMA solution with a concentration of 1.5 mg / mL, and store at -20°C. This concentration can effectively inhibit dead bacteria without affecting live bacteria.

[0058] Step S302, adding PMA solution: adding PMA solution to the microsphere-bacteria complex to obtain a mixed solution, the concentration of the microsphere-bacteria complex being 1.5 μg / mL, and stirring to mix.

[0059] Step S303, light-avoiding incubation: incubating the mixed solution at room temperature under light-avoiding conditions for 5 minutes to allow PMA to invade the damaged cell membrane of the dead bacteria.

[0060] Step S304, light treatment: placing the mixed solution on ice, and irradiating it for 20 minutes at a distance of 15 cm from a 500 W halogen lamp. The irradiation causes free PMA to photolyze into hydroxylamine, while PMA combined with the DNA of the dead bacteria forms a covalent bond, thereby inhibiting the amplification of the DNA of the dead bacteria.

[0061] Principle: PMA is a photoactivated dye that only enters the dead bacteria with damaged membranes, and is crosslinked with DNA after irradiation, so that only the DNA of the live bacteria is amplified in the detection.

[0062] Step S4, DNA extraction: extracting the bacterial DNA from the microsphere-bacteria complex.

[0063] This step is to release and purify high-quality DNA from the bacteria-microsphere complex subjected to immunocapture and PMA treatment, to provide a reliable template for subsequent real-time quantitative PCR detection.

[0064] Step S401, pretreatment and resuspension of the microsphere-bacteria complex.

[0065] A DNA extraction kit (QIAamp DNA Mini Kit produced by Qiagen, Germany) is used to extract the DNA.

[0066] At an ambient temperature of 4°C, 200 μL of pre-cooled PBS buffer is added to the mixed solution after PMA treatment to obtain a sample containing the microsphere-bacteria complex to be extracted; the sample to be extracted is oscillated at a medium speed (2500 rpm) for 30 seconds using a vortex oscillator to ensure that the microsphere-bacteria complex is completely resuspended, and the solution is observed to be uniformly turbid without visible clumps. Temperature: 4°C is maintained to prevent DNA degradation. Resuspension time: 30 seconds to avoid cell damage caused by too long time.

[0067] Step S402, enzymatic lysis process.

[0068] 20 μL of a protease K solution is added to the sample to be extracted, and the concentration of the protease K is 200 μg / mL; the sample to be extracted is incubated in a water bath at 56°C for 30 minutes, and is gently inverted and mixed once every 10 minutes during the incubation; the solution is observed to change from turbid to clear, indicating that the cell lysis is complete.

[0069] Step S403, binding and column purification.

[0070] After lysis, 200 μL of AL buffer is added to the sample and shaken well. The sample is incubated at 70°C for 10 minutes until it becomes clear and viscous. 200 μL of absolute ethanol (96-100%) is added immediately to prevent DNA precipitation, and the sample is shaken well. The sample is transferred to a QIAamp spin column, which is centrifuged at 8000 g for 1 minute. DNA molecules are specifically bound to the surface of the silica-based filter membrane of the QIAamp spin column through hydrophobic interaction and ion exchange. The solid after centrifugation is removed.

[0071] AL buffer: The main component is chaotropic salt (such as guanidine isothiocyanate), which can destroy the cell membrane structure, denature proteins, release DNA, and protect it from degradation.

[0072] Proteinase K: a hydrolase that specifically degrades nuclear proteins and impurities, improving DNA purity.

[0073] QIAamp spin column: a silica-based filter membrane with a specific pore size that selectively binds DNA in the presence of chaotropic salt.

[0074] Step S404, washing and elution; 1. The solid after centrifugation is washed. First washing: 500 μL of AW1 buffer is added, and the sample is centrifuged at 8000 g for 1 minute to remove residual proteins, enzyme inhibitors, and other polar impurities, obtaining a DNA-membrane complex. Second washing: 500 μL of AW2 buffer is added to the DNA-membrane complex, and the sample is centrifuged at 14000 g for 3 minutes to completely remove salt and organic solvent residues, avoiding PCR inhibition, obtaining a high-purity DNA-membrane complex. Higher centrifugal force ensures complete removal of ethanol, preventing subsequent reactions from being affected. Then, the sample is air-dried by centrifugation at 14000 g for 1 minute to completely remove residual ethanol, obtaining a high-purity DNA-membrane complex.

[0075] AW1 buffer: a high-salt solution that removes residual proteins and enzyme inhibitors.

[0076] AW2 buffer: an ethanol-based solution that completely removes salt and organic impurities.

[0077] 2. The high-purity DNA-membrane complex is transferred from the spin column to a 2 mL collection tube. 50 μL of AE buffer preheated to 70°C is added to the collection tube to enhance DNA solubility. The sample is allowed to stand at room temperature for 5 minutes to allow the buffer to fully soak the membrane. The collection tube is centrifuged at 8000 g for 1 minute to collect the eluate, obtaining a high-purity genomic DNA solution.

[0078] AE buffer: 10 mM Tris-Cl, pH 8.5, a low-ionic-strength buffer that promotes the dissociation of DNA from the membrane.

[0079] Step S5, Real-time quantitative PCR detection: Real-time quantitative PCR is performed on the nuc gene to quantify the concentration of live S. aureus by standard curve; the high-purity genomic DNA solution obtained after PMA treatment and DNA extraction of the milk sample to be tested is subjected to real-time quantitative PCR detection.

[0080] The method is based on the SYBR Green fluorescent dye method, and the bacterial concentration is calculated by the standard curve method, and the detection lower limit is 10 CFU / mL, the linear range is 10~10 8 CFU / mL, R²>0.99.

[0081] Step S501, primer design: Target gene: nuc gene, which is a specific marker of S. aureus.

[0082] Primer sequence: upstream primer: 5'-GCGATTGATGGTGATACGGTT-3', downstream primer: 5'-AGCCAAGCCTTGACGAACTAAAGC-3'.

[0083] Amplification fragment: 269bp, verified by BLAST to have species specificity (no significant homology with non-target strains such as E. coli and streptococcus).

[0084] The primers are optimized by Oligo 7 software to avoid dimers and hairpin structures, and the Tm value is calculated as 60°C.

[0085] Step S502, reaction system: total volume 20μL, including 10μL of SYBR Green fluorescent dye premix (2 times concentration), 0.5μL of upstream primer, 0.5μL of downstream primer, 5μL of template DNA, 4μL of nuclease-free water.

[0086] Template DNA, which is derived from the extracted DNA after PMA treatment, has a concentration of 1~10ng / μL.

[0087] Each batch of reaction sets a negative control (no template water) and a positive control (S. aureus standard), to monitor contamination and reagent effectiveness.

[0088] Step S503, real-time quantitative PCR program: Roche LightCycler 480 instrument is used, and the program parameters are as follows: Pre-denaturation: 95℃ for 10 minutes, to activate the polymerase and completely denature the DNA.

[0089] Amplification cycle (45 cycles): denaturation: 95°C, 10 seconds, 4.4°C / s, to ensure complete melting of DNA. Annealing: 60°C, 5 seconds, 2.2°C / s, optimized based on primer Tm value, balance specificity and efficiency. Extension: 72°C, 30 seconds, 4.4°C / s, to meet the synthesis requirements of 269 bp fragments.

[0090] Melting curve analysis: heating from 95°C to 58°C at a rate of 0.11°C / s, continuously collecting fluorescence signals. Purpose: to verify the specificity of the amplification product, and a single peak indicates no primer dimer or non-specific amplification.

[0091] Step S504, quantitative analysis: Standard curve preparation: standard curve was prepared with S. aureus standard, gradient dilution was 10-10 8 CFU / mL series concentration; the log value (log10 CFU / mL) of the standard concentration was used as the abscissa, and the threshold cycle number (Ct value) was used as the ordinate, linear regression was performed, R²> 0.99, and the slope was between -3.1 and -3.6 (corresponding to amplification efficiency 90%-110%).

[0092] Step S505, sample concentration calculation: the Ct value of the sample to be tested was substituted into the standard curve equation to calculate the concentration value. Judgment criteria: Ct value < 35 is considered positive; Ct value ≥ 35 or no detectable signal is considered negative.

[0093] Data verification: each group of experiments was repeated three times, and the average value was taken, with a coefficient of variation (CV) < 5% considered acceptable. Automatic analysis using instrument built-in software (such as LightCycler 480 Software) to avoid human error.

[0094] Each concentration point was detected three times, and the Ct value (threshold cycle number) was recorded. The data is shown in Table 1 below.

[0095] Table 1 is a summary table of detection data.

[0096]

[0097] Standard curve establishment: The log concentration (log10 CFU / mL) was used as the abscissa (X), and the Ct average value was used as the ordinate (Y) for linear regression analysis.

[0098] Regression equation: Y = -3.32X + 38.95 (slope = -3.32, intercept = 38.95).

[0099] Linear correlation coefficient: R² = 0.998, R²> 0.99, which meets the requirements.

[0100] Efficiency calculation: Efficiency = 10-1 / slope = 10-1 / -3.32 ≈ 100.3%, in the range of 90%-110%.

[0101] The curve is based on Roche LightCycler 480 instrument analysis, with CV <5% for each concentration point, indicating good data reproducibility. The slope of -3.32 falls within the range of -3.1 to -3.6, ensuring quantitative accuracy.

[0102] A specific example of sample concentration calculation is as follows: Assuming that the milk sample to be tested is treated with PMA and DNA extraction, real-time quantitative PCR detection is performed to obtain the Ct value. The following demonstrates how to calculate the concentration using the above standard curve: Sample detection data: Sample number: Milk-Sample-001.

[0103] Ct value result: The three repeated detection values are 26.8, 27.1, and 26.9, with an average Ct of 26.93 and a CV of 0.56%.

[0104] Concentration calculation steps: 1. Substitute the average Ct value (Y = 26.93) into the standard curve equation: Y = -3.32X + 38.95.

[0105] 2. Solve the equation: X = (Y - 38.95) / -3.32 ≈ 3.62 (log10 CFU / mL).

[0106] 3. Convert to linear concentration: Concentration = 10 X = 10 3.62 ≈ 4170 CFU / mL.

[0107] Judgment result: The average Ct value = 26.93 < 35, so it is judged to be positive. The melting curve analysis shows a single peak, confirming that the amplification product is highly specific and there is no contamination or non-specific amplification.

[0108] Practical significance: The concentration of active Staphylococcus aureus in this sample is 4.17 x 10 3 CFU / mL, which is lower than the common food safety limit (10 4 CFU / mL), and can be considered as low risk.

[0109] To verify the method of the present application, the following experiments were conducted: to evaluate the accuracy and practicality of the method in commercially available milk samples, and to compare it with the standard culture method.

[0110] Experimental steps: 1. Sample collection: 50 samples of commercially available pasteurized milk (covering different brands, batches) were collected, transported at 4℃ and processed within 2 hours.

[0111] Parallel sampling: Each sample was divided into two parts, one for this protocol and one for the standard culture method.

[0112] 2. Detection procedure: Group using this protocol: Sample pretreatment: 10 mL of milk was centrifuged at 3000 g for 10 minutes to remove fat, and the precipitate was resuspended in 1 mL of PBS buffer.

[0113] PMA treatment, DNA extraction, and real-time quantitative PCR detection were performed according to the sensitivity test procedure.

[0114] Standard culture method group: Spread method: 0.1 mL of the appropriate dilution was spread on CHROMagar S. aureus culture medium and incubated at 37℃ for 24-48 hours.

[0115] Confirmation and identification: coagulase test and PCR verification (nuc gene) were performed on typical colonies.

[0116] 3. Data analysis: Accuracy calculation: True positive (TP): Both methods were positive.

[0117] True negative (TN): Both methods were negative.

[0118] Accuracy = (TP + TN) / total sample number × 100%.

[0119] Time efficiency record: Total time from sample processing to result output.

[0120] 4. Expected results: Accuracy ≥ 95%. PMA-real-time quantitative PCR takes about 6 hours, and the culture method takes 24-48 hours.

[0121] 5. Quality control: Positive control (spiked sample) and negative control (sterile PBS) were set up for each batch.

[0122] Data statistics: Kappa coefficient was used to evaluate consistency, Kappa > 0.8.

[0123] Comparison with actual samples: Among the 50 samples of commercially available milk, the accuracy of this method and the standard culture method was 95%, and the time was shortened to 6 hours.

[0124] Sensitivity test: Staphylococcus aureus was added to sterile milk in serial dilutions (10-106 CFU / mL), detected as above. The results show a detection limit of 10 CFU / mL, a linear range of 10- 10 6 CFU / mL (R2= 0.998).

[0125] Specificity test: common bacteria such as E. coli, Salmonella, etc. were detected, only Staphylococcus aureus was positive, indicating that the primer has high specificity.

[0126] Live and dead bacteria differentiation: mix live bacteria with heat-killed bacteria (90°C, 20 minutes) at 1:1, PMA-real-time quantitative PCR only quantifies live bacteria, and the inhibition rate of dead bacteria is >99%.

[0127] In addition, it should be understood that although the present specification is described in terms of embodiments, not every embodiment contains only one independent technical solution, and the description of the specification is only for the sake of clarity, and those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can be appropriately combined to form other embodiments that those skilled in the art can understand.

Claims

1. A rapid and accurate detection method of Staphylococcus aureus in milk, characterized in that, The method comprises the following steps: Step S1, milk sample pretreatment: dilute and remove the upper lipid of the milk sample; Step S2, immunocapture enrichment of Staphylococcus aureus: co-incubate IgG cross-linked agarose microspheres with the milk sample, enrich the bacteria by specific binding of the surface protein A of Staphylococcus aureus to the Fc fragment of IgG, and obtain a microsphere-bacterium complex; Step S3, PMA treatment: add PMA dye to the microsphere-bacterium complex, carry out light shielding incubation and light irradiation to inhibit DNA amplification of dead bacteria, and obtain a mixed solution containing the microsphere-bacterium complex; Step S4, DNA extraction: extract bacterial DNA from the microsphere-bacterium complex in the mixed solution, and obtain a high-purity genomic DNA solution; Step S5, real-time quantitative PCR detection: perform real-time quantitative PCR on the nuc gene, and quantitatively determine the concentration of active Staphylococcus aureus by a standard curve; the high-purity genomic DNA solution obtained after PMA treatment and DNA extraction of the milk sample to be detected is subjected to real-time quantitative PCR detection.

2. The method for rapid and accurate detection of Staphylococcus aureus in milk according to claim 1, characterized in that, Step S1 comprises the following steps: Step S101, milk sample collection: collect 10 mL of the milk sample to be detected, store at 4°C, and centrifuge to remove the upper lipid; Step S102, dilution: dilute the milk sample by 1:10 with sterile phosphate buffer to reduce the matrix effect; Step S103, filtration: the milk sample is filtered through a 0.45 μm filter membrane to remove large particle impurities, and 20 mL of the milk sample is taken for standby.

3. The method for rapid and accurate detection of S. aureus in milk according to claim 2, characterized in that, Step S2 comprises the following steps: Step S201, preparation of agarose microspheres: 1, preparation of an aqueous phase: add 2% agarose by mass fraction to physiological saline containing 0.9% NaCl, microwave heat to clarify, and place in a 90°C water bath to remove air bubbles to obtain an aqueous phase; 2, preparation of an oil phase: add 3% surfactant Span80 by volume fraction to liquid paraffin, heat to 70°C and stir uniformly to obtain an oil phase; 3, emulsification: mix the aqueous phase and the oil phase at a volume ratio of 1:6 in a 60°C water bath at 4000 rpm for 10 minutes; after emulsification, cool at 4°C for 10 minutes to solidify the microspheres; 4. Washing: centrifugal washing with deionized water, 20% ethanol and anhydrous ethanol in turn, and finally filtering through a 100 μm screen to collect agarose microspheres of 50-100 μm in diameter, and storing in 50% ethanol to obtain an agarose microsphere suspension with a concentration of about 10 6 -10 7 microspheres / mL. Step S202, IgG cross-linking, to obtain an IgG-agarose microsphere suspension: 1, take 2.5 mL of the agarose microsphere suspension, wash twice with deionized water to remove ethanol residues; then add to 0.5 mol / L sodium carbonate solution containing 5% epichlorohydrin, and oscillate for 2 hours to activate the epoxy groups on the surface of the microspheres; 2, mix the sodium carbonate solution containing the agarose microspheres with the purified IgG solution, oscillate for 24 hours to allow the IgG to be cross-linked to the microspheres by covalent bonds, and centrifuge to remove the supernatant to obtain IgG-agarose microspheres; 3, add the IgG-agarose microspheres to the sodium carbonate solution containing 8.5% ethanolamine, remove non-specific adsorption, and obtain an IgG-agarose microsphere suspension after stirring, which is reserved for standby; Step S203, capture incubation, to obtain a microsphere-bacterium complex: Take 1 mL of IgG-agarose microspheres suspension and mix with 20 mL of pretreated milk sample, incubate at 37°C on a shaker at 100 rpm for 1 hour to allow specific binding of S. aureus to IgG; After incubation, centrifuge at 1000 g for 5 minutes to collect the microsphere-bacteria complex, wash with PBS buffer for 3 times to remove unbound components.

4. The method for rapid and accurate detection of Staphylococcus aureus in milk according to claim 1 or 3, characterized in that, Step S3 includes the following steps: Step S301, PMA solution preparation: dissolve PMA powder with sterile deionized water to prepare a PMA solution with a concentration of 1.5 mg / mL, and store at -20°C; Step S302, PMA solution addition: add PMA solution to the microsphere-bacteria complex to obtain a mixed solution, with a concentration of 1.5 μg / mL of microsphere-bacteria complex, and mix well; Step S303, incubation in the dark: incubate the mixed solution in the dark at room temperature for 5 minutes to allow PMA to invade the damaged cell membrane of dead bacteria; Step S304, light treatment: place the mixed solution on ice, 15 cm away from a 500W halogen lamp, and irradiate for 20 minutes; light irradiation causes free PMA to photolyze into hydroxylamine, while PMA bound to dead bacterial DNA forms a covalent bond, inhibiting the amplification of dead bacterial DNA.

5. The method for rapid and accurate detection of S. aureus in milk according to claim 4, characterized in that, Step S4 includes the following steps: Step S401, pretreatment and resuspension of microsphere-bacteria complex: use a DNA extraction kit to extract DNA; add 200 μL of pre-cooled PBS buffer to the mixed solution after PMA treatment at an ambient temperature of 4°C to obtain a sample containing microsphere-bacteria complex; use a vortex shaker to oscillate the sample at medium speed for 30 seconds to ensure complete resuspension of the microsphere-bacteria complex, and observe that the solution is uniformly turbid without visible clumps; Step S402, enzymatic lysis process: add 20 μL of proteinase K solution to the sample to obtain a concentration of 200 μg / mL of proteinase K; incubate the sample in a 56°C water bath for 30 minutes, gently invert and mix every 10 minutes; observe that the solution changes from turbid to clear, indicating complete cell lysis; Step S403, binding and column purification: add 200 μL of AL buffer to the lysed sample and shake well; incubate at 70°C for 10 minutes until the sample becomes clear and viscous; immediately shake well after adding 200 μL of anhydrous ethanol to prevent DNA precipitation; transfer the entire sample solution to a QIAamp centrifugal column, centrifuge at 8000 g for 1 minute, and the DNA molecules are specifically bound to the silicon-based filter membrane surface of the QIAamp centrifugal column through hydrophobic interaction and ion exchange, and then collect the solid after centrifugation; Step S404, washing and elution; 1. Washing the centrifuged solid; first washing: adding 500 μL of AW1 buffer, centrifugation condition is 8000g and centrifugation for 1 minute, obtaining DNA-membrane complex; second washing: adding 500 μL of AW2 buffer to the DNA-membrane complex, centrifugation condition is 14000g and centrifugation for 3 minutes, obtaining high-purity DNA-membrane complex; then empty centrifugation, centrifugation condition is 14000g and centrifugation for 1 minute, obtaining high-purity DNA-membrane complex; 2. Transferring the high-purity DNA-membrane complex from the centrifugal column to a collection tube with a volume of 2mL; adding 50 μL of AE buffer preheated to 70°C in the collection tube to enhance the solubility of DNA; standing at room temperature for 5 minutes to allow the buffer to fully soak the membrane; centrifuging the collection tube at 8000g for 1 minute to collect the eluent, obtaining a high-purity genomic DNA solution.

6. The method for rapid and accurate detection of S. aureus in milk according to claim 5, characterized in that, Step S5 includes the following steps: Step S501, primer design: Target gene: nuc gene, which is a specific marker of Staphylococcus aureus; Primer sequence: upstream primer: 5'-GCGATTGATGGTGATACGGTT-3', downstream primer: 5'-AGCCAAGCCTTGACGAACTAAAGC-3'; Amplification fragment: 269bp, verified by BLAST to have species specificity; Step S502, reaction system: total volume 20 μL, including 10 μL of 2-fold concentration SYBR Green fluorescent dye premix, 0.5 μL of upstream primer, 0.5 μL of downstream primer, 5 μL of template DNA and 4 μL of nuclease-free water; Template DNA is extracted DNA derived from PMA treatment, with a concentration of 1-10 ng / μL; Step S503, real-time quantitative PCR treatment; Step S504, quantitative analysis: Standard curve preparation: standard curve was prepared with S. aureus standard, gradient dilution was 10~10 8 CFU / mL series concentration; with the logarithmic value of standard concentration as the abscissa, the threshold cycle Ct value as the ordinate, linear regression was performed; Sample concentration calculation: substitute the Ct value of the sample to be tested into the standard curve equation to calculate the concentration value; determination standard: Ct value < 35 is considered positive; Ct value ≥ 35 or no detectable signal is considered negative.

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