Method for lysing foodborne pathogenic bacteria and application thereof

Abstract

The present application relates to the technical field of food safety, and particularly relates to a low-power water bath ultrasonic foodborne pathogenic bacteria lysis method and application thereof. The present application uses a low-power water bath ultrasonic machine, adds ABS small balls with a size of about 1 mm in a solution sample, uses ultrasonic to cause high-frequency vibration of the ABS small balls, and the local high sound pressure field and high-density cavitation field generated by the vibration can rapidly lyse bacteria. Meanwhile, the movement of the small balls can accelerate the rapid material exchange of the solution at different positions in the tube, so that 1 mL of the to-be-tested bacterial sample can be lysed within 3 minutes. The method provided by the present application has high lysis efficiency and short time consumption. When applied to foodborne pathogenic bacteria detection, nucleic acid purification and enrichment of the lysis liquid are not needed, the time and labor cost required for nucleic acid detection can be greatly shortened, and a new method of efficient, economical and practical bacterial lysis and nucleic acid extraction is provided for the field of food safety detection.

Description

Technical Field

[0001] This invention relates to the field of food safety technology, and in particular to a method for lysing foodborne pathogens using low-power water bath ultrasound and its application. Background Technology

[0002] In the field of food safety, rapid and accurate detection of foodborne pathogens is a crucial step in safeguarding public health. According to national standards such as GB 4789.4—2016, the sampling stage for foodborne pathogen detection often requires pre-enrichment and enrichment treatment to ensure that the bacterial count in the sample reaches the detection limit of the method. This process typically takes at least 8 to 18 hours. However, traditional detection methods, such as culture methods and PCR gel electrophoresis, while providing accurate results, suffer from drawbacks such as long processing times, complex procedures, and high costs, making it difficult to meet the demands of modern food safety testing for speed and efficiency.

[0003] In recent years, with the rapid development of molecular biology techniques, qPCR (quantitative real-time PCR) has been widely used in the detection of foodborne pathogens due to its advantages such as high sensitivity, high specificity, and speed. qPCR achieves rapid quantitative detection of pathogens by directly detecting bacterial nucleic acids in samples. Currently, qPCR is considered the "gold standard" method for detecting various pathogens. However, to perform qPCR detection, high-quality, high-concentration nucleic acids must first be extracted from the sample. Bacterial lysis, as a key step in nucleic acid extraction, directly impacts the performance of the entire detection process due to its efficiency and cost.

[0004] Traditional bacterial lysis methods include chemical lysis, thermal lysis, and ultrasonic lysis. While chemical lysis is highly efficient, it involves complex procedures, requires additional nucleic acid purification steps, and incurs high reagent costs. It also demands specialized laboratory conditions and personnel, leading to additional laboratory, time, and labor costs for qPCR testing. Thermal lysis is relatively inefficient and prone to nucleic acid aerosol contamination. Ultrasonic lysis relies on high-powered ultrasonic equipment, and currently lacks effective methods for pathogen nucleic acid extraction. However, it typically requires expensive ultrasonic lysis equipment and lengthy lysis times, further increasing testing costs.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a method for lysing foodborne pathogens using low-power water bath ultrasound and its application.

[0007] Specifically, the technical solution of the present invention is as follows:

[0008] In a first aspect, the present invention provides a method for lysing foodborne pathogens, comprising: adding ABS microspheres to a solution sample of foodborne pathogens, and using ultrasound to induce high-frequency vibration of the ABS microspheres, thereby achieving lysis of the bacteria; the particle size of the ABS microspheres is 1.0±0.2 mm; the ultrasound parameters are 0.12±0.01 W / mL and 40±3 kHz.

[0009] Preferably, the ultrasonic treatment time is 2.5 ± 0.1 min.

[0010] Preferably, 0.2 ± 0.01 g of ABS microspheres are added to each 1 mL solution sample of foodborne pathogens.

[0011] Preferably, the solution sample of foodborne pathogens is placed in a centrifuge tube; the angle between the centrifuge tube and the ultrasonic transducer during the ultrasonic process is 90±5°.

[0012] Preferably, the solution sample of foodborne pathogens is a cultured or uncultured food sample to be tested.

[0013] Preferably, the food sample to be tested is from at least one of the following: chicken offal, milk, coconut milk, chicken legs, wastewater from the chicken counter, orange juice, and apple juice.

[0014] Preferably, the bacterial content in the solution sample of foodborne pathogens is <6.8 CFU / mL, 6.8~13.6 CFU / mL, 13.6~13600 CFU / mL, or >13600 CFU / mL.

[0015] Preferably, foodborne pathogens include Gram-positive bacteria and / or Gram-negative bacteria.

[0016] Preferably, Gram-positive bacteria include Bacillus cereus ATCC 14579; and / or, Gram-negative bacteria include Escherichia coli O157:H7 ATCC 43888 or Salmonella typhimurium ATCC 14028.

[0017] Secondly, the present invention provides the application of the lysis method of the foodborne pathogens in the detection of foodborne pathogens for non-disease diagnostic purposes.

[0018] Beneficial effects:

[0019] This invention provides a low-power water bath ultrasonic lysis method for foodborne pathogens and its application. Using a low-power water bath ultrasonic machine, ABS microspheres approximately 1 mm in size are added to the solution sample. Ultrasonic waves induce high-frequency vibrations in the ABS microspheres, generating a localized high-sound-pressure field and a high-density cavitation field that rapidly lyses the bacteria. Simultaneously, the movement of the microspheres accelerates rapid material exchange within the solution at different locations in the tube, allowing 1 mL of the test bacterial sample to be lysed within 3 minutes. The method provided by this invention offers high lysis efficiency and short processing time. When applied to the detection of foodborne pathogens, it eliminates the need for nucleic acid purification of the lysate (no chemical reagents involved) and enrichment of the lysate (high detection limit), significantly reducing the time and labor costs required for nucleic acid detection. This provides a highly efficient, economical, and practical new method for bacterial lysis and nucleic acid extraction in the field of food safety testing. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in this invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be described below.

[0021] Figure 1 To verify the feasibility of the ultrasonic grinding method using beads, Figure A shows a schematic diagram of ultrasonic grinding of beads in a water bath. The sample tube should be perpendicular to the direction of sound wave propagation to facilitate the absorption of ultrasonic energy by the grinding beads. Figure B shows 10 9 The difference in nucleic acid concentration of CFU / mL bacteria between conventional sonication (without grinding beads) and sonication with beads.

[0022] Figure 2 To compare the lysis performance of the ultrasonic bead grinding method with other methods, Figure A shows the differences between ultrasonic bead grinding and other physical bacterial lysis methods. Significant differences were observed between ultrasonic bead grinding and thermal lysis, repeated freeze-thaw cycles, and conventional ultrasound. Figure B shows the spectral data after ultrasonic bead grinding, thermal lysis, repeated freeze-thaw cycles, and conventional ultrasound, revealing a nucleic acid characteristic peak at 260 nm, confirming that this method can achieve bacterial nucleic acid extraction.

[0023] Figure 3 The images show TEM characterization of the ultrasonic bead grinding method. Figures A, B, C, and D illustrate the TEM characterization of the original bacteria, conventional ultrasound, thermal lysis, and ultrasonic bead grinding, respectively. The images show a change in bacterial morphology from intact to fragmented, indicating that conventional ultrasound and thermal lysis can only partially lyse the bacteria, while ultrasonic bead grinding can completely lyse them. Figure E is a schematic diagram of the lysis effects of the four methods. Figure F shows the size of the bacterial fragments calculated using software; the statistical data is consistent with the above results.

[0024] Figure 4For the bead parameter optimization experiment, Figure A shows that 1 mm beads have the highest nucleic acid extraction efficiency. Figure B shows that among different bead materials, although the differences between materials are not significant, ABS plastic has the highest nucleic acid extraction efficiency and the lowest cost. Figure C shows that, in terms of dosage, 0.2 g of ABS beads has the highest nucleic acid extraction efficiency. Figure D shows whether the sample tube is perpendicular or parallel to the ultrasonic transducer for the best effect; the result is that the sample tube perpendicular to the ultrasonic transducer has the best effect.

[0025] Figure 5 This experiment tested the lysis performance of bacteria using ultrasonic grinding. Figure A shows the lysis efficiency of three bacteria: Bacillus cereus (59.82%), Escherichia coli O157:H7 (66.53%), and Salmonella (84.10%). Figure B shows the optimization of lysis time; over 80% lysis efficiency can be achieved in 150 seconds. Figure C compares the lysis efficiency of different lysis methods; the lysis efficiency of ultrasonic bead grinding is second only to that of commercial kits.

[0026] Figure 6 Figure A shows the amplification curves and standard curves for qPCR detection after ultrasonic grinding. Figure A shows the amplification curves for qPCR amplification after serial dilution. Figure B shows the standard curve with a linearity of 0.9946.

[0027] Figure 7 To illustrate the application of the method of this invention to actual sample testing, Figure A shows the positive rates of different samples under different bacterial addition conditions. Figure B shows the Ct values ​​of qPCR for different samples under the same bacterial concentration.

[0028] Figure 8 Figures show the results of the finite element simulation analysis. Figure A shows the force analysis of a single bead in a water bath ultrasonic bath. Figure B shows the interaction of multiple beads in a water bath ultrasonic bath. Figure C shows the sound velocity and sound pressure distribution from a single bead to multiple beads. Figure D shows the total sound pressure distribution inside beads of different sizes. Figures E, F, G, and H show the sound velocity and flow velocity fields when the beads are not vibrating and when the beads are vibrating. Detailed Implementation

[0029] This invention proposes a low-power water bath ultrasonic lysis method for foodborne pathogens, aiming to solve the problems of high cost and low efficiency in existing bacterial lysis methods. This method involves adding microspheres to the sample solution and using a commonly used low-power water bath ultrasonic cleaner for ultrasonic treatment, achieving rapid and effective lysis of rod-shaped Gram-positive and Gram-negative bacteria. This method not only simplifies the experimental procedure and avoids expensive equipment and reagent costs, but also significantly shortens the time for bacterial lysis and nucleic acid extraction, improving detection efficiency.

[0030] Specifically, the low-power water bath ultrasonic bacterial lysis method provided by this invention can complete the lysis of bacteria in 1 mL of sample solution within 3 minutes. qPCR performed using this method achieves a detection limit of 13.6 CFU / mL, significantly higher than the detection sensitivity of traditional methods. Since no lysis buffer is added, this method eliminates the need for nucleic acid purification steps, and the high detection limit also eliminates the need for nucleic acid enrichment, greatly reducing the time and labor costs required for nucleic acid detection. This method only requires a commonly used low-power (0.12 W / mL, 40±3 kHz) water bath ultrasonic cleaner (costing less than one-tenth the price of a laboratory ultrasonic cell disruptor) and 1 mm ABS beads, significantly reducing the cost of bacterial lysis and nucleic acid extraction. This provides a highly efficient, economical, and practical new method for bacterial lysis and nucleic acid extraction in the field of food safety testing.

[0031] The research process of this invention will be described in detail below.

[0032] This invention proposes a novel bacterial lysis method using a low-power water bath sonicator. 1mm ABS microspheres are added to the solution sample. Sonication causes the ABS microspheres to vibrate at high frequency. The resulting localized high sound pressure field and high-density cavitation field rapidly lyse the bacteria. Simultaneously, the movement of the microspheres accelerates rapid mass exchange within the solution at different locations in the tube, allowing 1mL of the test bacterial sample to be lysed within 3 minutes. During the research, this invention optimized the size, material, and quantity of the sonication microspheres, the placement of the centrifuge tube, the sonication time, and the types of effective bacteria. The lysis efficiency of different lysis methods was compared, demonstrating the effectiveness of this method. Furthermore, using Salmonella as an example, this invention verified the effectiveness of this method for detecting foodborne pathogens using qPCR, determined the limit of detection, and demonstrated the feasibility of applying this method to the field of foodborne pathogen detection using real samples.

[0033] First, this invention combines low-power water bath ultrasound with ABS microspheres to achieve lysis. It first uses a low-power water bath ultrasound machine under specific conditions (ultrasound power 0.12 W / mL, frequency 40±3KHz) to combine 1mm ABS microspheres (the microspheres are made of acrylonitrile-butadiene-styrene copolymer, with a diameter of 1mm, and the amount used in a commonly used 1.5mL centrifuge tube in the laboratory is preferably 0.2±0.01 g). The local high sound pressure field and high density cavitation field generated by high-frequency vibration achieve rapid bacterial lysis.

[0034] Secondly, the present invention optimizes the ultrasonic microspheres inside the tube, specifically by optimizing the size (diameter approximately 1 mm), material (acrylonitrile-butadiene-styrene copolymer), and dosage (0.2 ± 0.01 g in a commonly used 1.5 mL centrifuge tube in the laboratory). These optimizations play a crucial role in improving pyrolysis efficiency and ensuring pyrolysis effect, thus guaranteeing the realization of the technical effect of the present invention.

[0035] Furthermore, this invention optimizes and controls the operating procedures and conditions. First, the placement of the centrifuge tubes and the sonication time are optimized: the placement of the centrifuge tubes in the sonication equipment (the centrifuge tubes should be perpendicular to the ultrasonic transducer) and the sonication time (2.5±0.1 min) significantly affect the lysis effect of this invention. The lysis procedure is also standardized: sample preparation (according to national standard GB 4789.4—2016), taking 1 mL of sample, adding 0.2±0.01 g of 1 mm diameter ABS microspheres, sonicating for 3 minutes, and then aspirating the supernatant sample solution for qPCR detection. The entire lysis procedure is preferably implemented according to standardized operating steps to ensure the reproducibility and consistency of the technology.

[0036] Furthermore, the application effect of the scheme was verified by data. First, the lysis efficiency was compared and verified: by comparing the lysis efficiency data of different lysis methods (such as chemical lysis, thermal lysis, traditional ultrasonic methods, etc.), it was confirmed that the scheme provided by the present invention has significant superiority in these comparisons. Then, the limit of detection was verified using qPCR: the effectiveness of this method in the detection of foodborne pathogens (such as Salmonella) was verified using qPCR technology. For specific qPCR experimental design, operation steps, result analysis, and the determination data of the limit of detection (13.6 CFU / mL), please refer to the description in the examples. Since the present invention claims protection for the application of the lysis method of foodborne pathogens in the detection of foodborne pathogens for non-disease diagnostic purposes, the qPCR-related methods disclosed in the examples or the undisclosed content related to the detection of foodborne pathogens using the lysis method of foodborne pathogens also fall within the protection scope of the present invention. The present invention also verified the feasibility of its application to real samples: the feasibility of this method in the field of foodborne pathogen detection was verified by real food samples. Based on experimental data and analysis results, it was proved that the method of the present invention has reliability and practicality in practical applications.

[0037] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0038] The endpoints and any values ​​of the ranges disclosed in this specification are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0039] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "specific implementation," or "some specific implementations," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0040] In the embodiments provided in this specification, unless specific techniques or conditions are specified, the techniques or conditions described in the literature in this field, or the product instructions, shall be followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0041] In the following examples, the water bath ultrasonic cleaner KQ118 (40kHz, 70W, 600mL) was purchased from Kunshan Ultrasonic Instrument Co., Ltd. (Kunshan, China). qPCR analysis was performed using a Thermo Fisher Scientific Applied Biosystems QuantStudio 3 qPCR instrument. Nucleic acid concentrations were determined using a Thermo Fisher Scientific nanoDrop one C ultra-micro UV-Vis spectrophotometer. Homogenizer bags and a BagMixer 400CC were purchased from Interscience. Primers used for qPCR were synthesized by Shanghai Sangon Biotech Co., Ltd. The qPCR master mix used was Taq Pro Universal SYBR qPCR Master Mix, from Nanjing Novizan Biotechnology Co., Ltd. The commercial kit used in the control experiments was the Magnetic Universal Genomic DNA Kit purchased from Tiangen Biotech Co., Ltd. The temperature sensor was a TM-902C K-type thermocouple, purchased from Taizhou Nuowei Electrical Equipment Co., Ltd. No other materials or instruments had special requirements.

[0042] The bacterial strains used in the following examples included *Escherichia coli* O157:H7 CMCC 44102, *Bacillus cereus* ATCC 14579, and *Salmonella typhimurium* ATCC 14028. The bacteria were cultured using the Luria-Bertani (LB) broth method. Pure cultures of these bacteria were first inoculated into LB broth and incubated at 37°C for 16 hours to ensure optimal growth (this method allows the bacteria to proliferate to concentrations suitable for further experiments).

[0043] In the following examples, DNA concentrations are expressed as mean ± standard deviation (SD). Each experiment was performed at least five times. Significance analysis was performed using t-tests. A p-value less than 0.05 was considered statistically significant. Multiphysics simulations were performed using COMSOL 6.2 software. 3D printing modeling software was Solidworks 2024. Ct values ​​were calculated using a thresholding method, i.e., adding 10 standard deviations to the mean of the signal values ​​calculated in Design & Analysis Software 2.7.0 for the first ten cycles. ImageJ software was used to analyze the area of ​​transmission electron microscopy (TEM) characterization images. MATLAB R2024a and MATLAB cftool were used to perform linear regression analysis. Student's t-test was used in GraphPad software for significance analysis.

[0044] In the following examples, the supernatant of the bacterial extract was used to measure nucleic acid concentration in the subsequent proof-of-concept ultrasonic grinding experiment. 0.25 g of 1 mm diameter ABS beads were added to a 1.5 mL centrifuge tube and mixed with 1 mL of bacterial solution to be lysed. The 1.5 mL centrifuge tube was placed vertically on a low-power water bath ultrasonic cleaner using a 3D-printed holder. After 3 minutes of ultrasonic bead grinding and lysis, the supernatant was aspirated and centrifuged at 12,000 rpm to remove impurities, or directly subjected to PCR amplification. Five parallel experiments were performed for each concentration gradient. The lysis efficiency was determined by the absorbance of the bacterial solution at 600 nm, as A600 is directly related to the bacterial concentration (57, 58). The lysis efficiency E can be calculated using the following formula: E = (1 - A600_After) / A600_Before, where A600_After and A600_Before are the A600 values ​​before and after lysis, respectively.

[0045] In the following examples, ultrasonic bead grinding was used to replace the traditional nucleic acid extraction step in the kit. 10 8 CFU / mL bacterial solutions were dissolved in ultrapure water and lysed. The supernatant was serially diluted and detected by qPCR. After sonication with beads, serial dilutions were performed. qPCR amplification and signal reading were performed using different concentrations of extracted nucleic acids. For comparison with thermal lysis, the original bacterial solution was diluted with ddH2O, then mixed with grinding beads for 3 minutes, and the supernatant was used for qPCR amplification and detection. A 20 μL PCR amplification kit contained 10 μL of master mix, 2 μL of template, 0.8 μL of each primer, and 7.2 μL of ddH2O. The two-step PCR amplification program included preheating at 95°C for 30 seconds, followed by 60 cycles of denaturation at 95°C for 10 seconds, and annealing and extension at 60°C. Melting curve experiments were performed after the qPCR detection experiments to eliminate interference from non-specific amplification.

[0046] In the following examples, bacterial counts were determined using the LB agar plate method. After incubation at 37°C for 12 hours, colonies were counted to assess the bacterial load in the samples. First, Salmonella solution (1.36 × 10⁻⁶) was prepared. 8 (CFU / mL) was added to the food sample. The standard method GB 4789.4-2016 was strictly followed to ensure the integrity and reliability of the samples used in this study. In short, 25 mL of the food sample was first mixed with 225 mL of sterile phosphate-buffered saline (PBS) and homogenized for 2 minutes. Then, 0.25 g of ABS beads was mixed with 1 mL of the sample solution, sonicated for 3 minutes, and serially diluted to 1.36 × 10⁻⁶. 2 Up to 1.36 × 105 The concentration of CFU / mL was determined. Finally, bacterial nucleic acid was detected using qPCR. This invention selected chicken leg meat purchased from a supermarket, wastewater collected from the chicken counter, and chicken offal as poultry samples. Milk, coconut juice, orange juice, and apple juice from the shelves were also purchased as beverage samples.

[0047] Example 1

[0048] This embodiment provides a method for lysing foodborne pathogens. 1 mL of a solution sample of foodborne pathogens (according to national standard GB 4789.4—2016) is added to a 1.5 mL centrifuge tube, followed by 0.2 g of ABS microspheres with a particle size of 1 mm. A low-power water bath sonicator is used, with the ultrasonic parameters set to 0.12 W / mL and a frequency of 40±3 kHz, and the sonication is performed for 2.5 min.

[0049] Example 2

[0050] This embodiment describes the experimental content of the relevant research of Embodiment 1 in conjunction with the accompanying drawings.

[0051] The main principle of bacterial lysis is the repeated expansion and contraction of beads under the interaction of high and low pressure zones in ultrasound. When the beads contract, they pressurize the bacteria and the surrounding solution; when they expand, the pressure decreases, and cavitation occurs simultaneously. This cycle repeats, leading to rapid bacterial lysis and release of DNA. Ultimately, this method can improve the sensitivity of qPCR detection.

[0052] During grinding, the beads are added to centrifuge tubes. Under ultrasonic excitation, the beads vibrate at high frequency, and the resulting acoustic flow, cavitation, and convection effects work together to complete bacterial lysis and nucleic acid extraction in a large volume (1 mL) sample. Among them, the acoustic flow synergistic cavitation effect enables bacterial lysis, while the acoustic flow convection effect enables rapid material exchange, thereby improving lysis efficiency.

[0053] The process for detecting foodborne pathogens using ultrasonic bead grinding includes three steps: The first step is food sample preparation and pretreatment, including homogenization, filtration, centrifugation, and pre-enrichment. Specific procedures for this step should be performed according to the national standard GB 4789.1-2016, based on actual needs. The second step involves bacterial lysis and nucleic acid extraction. An appropriate amount of beads is added to a centrifuge tube, which is then placed vertically on an ultrasonic cleaner and sonicated for 3 minutes. The third step involves mixing the sample with a qPCR reaction system and performing qPCR detection. This method significantly improves the bacterial lysis efficiency of low-power ultrasound, reduces detection costs, and simplifies the detection process for foodborne pathogens.

[0054] The feasibility verification of the ultrasonic grinding method with added beads can be found in [link to documentation]. Figure 1Figure A shows a schematic diagram of ultrasonic grinding using a water bath. The sample tube should be perpendicular to the direction of sound wave propagation to facilitate the absorption of ultrasonic energy by the grinding beads (the sample tube and the ultrasonic transducer should be at a 90° angle to ensure the best and fastest pyrolysis). Figure B shows 10 9 The difference in nucleic acid concentration of CFU / mL bacteria between conventional sonication (without grinding beads) and sonication with beads.

[0055] A comparison of the pyrolysis performance of the method of this invention with other methods is shown in the figure. Figure 2 Figure A illustrates the differences between the ultrasonic bead grinding method and other physical bacterial lysis methods. Significant differences were observed between ultrasonic bead grinding and thermal lysis, repeated freeze-thaw cycles, and conventional ultrasound (the lysis efficiency after adding beads was significantly higher than thermal lysis, and significantly higher than ultrasound without beads at the same time and placement). Figure B shows the spectral data after ultrasonic bead grinding, thermal lysis, repeated freeze-thaw cycles, and conventional ultrasound. A characteristic nucleic acid peak at 260 nm is visible, confirming that this method can extract bacterial nucleic acids (the absorption peak of the extracted nucleic acid measured using a nano-drop shows a clear absorption peak at 260 nm, demonstrating the feasibility of nucleic acid extraction and the integrity of the nucleic acid chain).

[0056] TEM characterization image (see) Figure 3 Figures A, B, C, and D show TEM characterization images of the original bacteria, after conventional ultrasound, thermal lysis, and ultrasonic bead grinding, respectively. The images reveal a range of bacterial morphology from intact to fragmented, indicating that conventional ultrasound and thermal lysis can only partially lyse the bacteria, while ultrasonic bead grinding can completely lyse them. Figure E is a schematic diagram of the lysis effects of the four methods. Figure F shows the size of the bacterial fragments calculated using software; the statistical data is consistent with the above results.

[0057] In a low-power water bath ultrasonic bead milling method, the forces and motion of the beads during water bath ultrasonication are analyzed. In the solution, the beads are mainly subjected to acoustic radiation force, buoyancy, gravity, fluid resistance, and interaction forces between the beads. Analyzing the forces on a single bead, a single bead in ultrasonication experiences gravity, buoyancy, fluid resistance, and acoustic radiation force, while cavitation bubbles are generated on its surface due to friction. Analyzing the forces on multiple beads, in addition to the above forces, the beads are also subject to interaction forces between them, which convert ultrasonic energy into heat, potentially promoting cavitation. Multiple beads will generate more cavitation bubbles.

[0058] Finite element method (FEM) simulation analysis of the physical fields related to ultrasonic grinding revealed that the aggregation of multiple beads significantly increases the local sound pressure level. Furthermore, the magnitude of the local sound pressure level primarily depends on the size of the beads. Appropriate bead size and quantity provide a large adhesion area for cavitation, thereby significantly enhancing acoustic cavitation. Simultaneously, the increased local sound velocity and flow rate generated by the high-frequency vibration of the beads can also help improve nucleic acid extraction efficiency. (See...) Figure 8 .

[0059] To verify that the interaction between multiple beads increases local sound pressure, a two-dimensional finite element method was used to analyze the sound velocity and sound pressure distribution of 1 mm diameter beads in single, two, and four-bead groups under contracted conditions. The sound pressure distribution at the center of four beads of different sizes was also investigated. Simulation results show that the sound pressure is highest when four 1.5 mm diameter beads are clustered together.

[0060] Comparing the acoustic-fluid convection effects (sound velocity distribution and flow velocity distribution) of beads under vibration and non-vibration conditions, verification and simulation results show that: high gradient sound velocity is concentrated between beads; when beads vibrate at high frequencies, they drive the fluid to move at high speed, thereby achieving efficient material exchange and improving grinding efficiency.

[0061] See the experiment on bead parameter optimization. Figure 4 Figure A shows that 1 mm beads resulted in the highest nucleic acid extraction efficiency. Figure B shows that among different bead materials, although the differences between materials were not significant, ABS plastic had the highest nucleic acid extraction efficiency and the lowest cost. Therefore, ABS plastic was chosen as the material for grinding beads in subsequent experiments. Figure C shows that 0.2 g of ABS beads resulted in the highest nucleic acid extraction efficiency. Figure D shows whether the sample tube was perpendicular or parallel to the ultrasonic transducer for the best effect; the result was that the sample tube was perpendicular to the ultrasonic transducer for the best effect. The above experiments optimized and verified the placement of the centrifuge tube (an angle of 90° with the ultrasonic transducer was optimal), the material of the ultrasonic beads in the centrifuge tube (ABS beads were the best), the diameter of the ultrasonic beads in the centrifuge tube (1 mm was optimal), and the amount of ultrasonic beads used in a 1.5 mL centrifuge tube (0.2 ± 0.01 g was optimal).

[0062] In low-power water bath ultrasonic bead milling, in addition to the movement of the beads, bacteria are also subjected to non-uniform shear forces between the beads. Specifically, rod-shaped bacteria are subjected to non-uniform acoustic radiation forces (compressive and expansive forces from acoustic pressure waves). Simultaneously, as the bacteria approach the beads, they are also subjected to shear forces and rupture impact forces from cavitation bubbles (cavitation bubble impact). Other forces, such as fluid shear forces (compressive forces) and bacterial protein denaturation leading to disintegration, also promote bacterial lysis. In the ultrasonic field, the surface acceleration of the beads is tangential to the surface, which promotes friction between the bead surface and the solution, thus fostering cavitation. Analysis of the forces acting on the bacteria reveals that, like the beads, the bacteria are primarily subjected to gravity, buoyancy, fluid resistance, acoustic radiation forces, and bead pressure. Analysis of the surface velocity direction of the grinding beads under ultrasonic fields indicates that high surface acoustic velocities lead to greater cavitation effects, which contribute to bacterial lysis. Simulations of the acoustic velocity on the bacterial surface between two beads show that non-uniform acoustic velocities on the bacterial surface can lead to faster bacterial fragmentation.

[0063] Three different bacteria were selected: Bacillus cereus ATCC 14579 (Gram-positive), Escherichia coli O157:H7 ATCC 43888 (Gram-negative), and Salmonella typhimurium ATCC 14028 (Gram-negative). The lysis efficiency of the lysis method of this invention, commercial kits, 70°C water bath heating, and lysis buffers such as proteinase K and lysozyme were tested.

[0064] The results of the pyrolysis efficiency test are shown in Figure 5 Figure A shows the lysis efficiency of three bacteria: Bacillus cereus (59.82%), Escherichia coli O157:H7 (66.53%), and Salmonella (84.10%). (This invention demonstrates high lysis efficiency for these three common foodborne pathogens.) Figure B shows the optimized lysis time; over 80% lysis efficiency can be achieved in 150 seconds (time optimization: 150 seconds can complete lysis of 1 mL of 10...). 9 Lysis of Salmonella culture at CFU / mL). Figure C compares the lysis efficiency of different lysis methods; the lysis efficiency of ultrasonic bead milling is second only to that of the commercial kit (ultrasound + beads per 1 mL 10... 9 The lysis efficiency of CFU / mL Salmonella culture was comparable to that of commercial kits. However, sonication with microspheres only took 3 minutes, while commercial kits required more than 15 minutes.

[0065] After lysing the above three bacteria, qPCR melting curve analysis was performed. The results showed that the correct sequences could be amplified, verifying the feasibility of qPCR detection after sonication. (Using the method of this invention for qPCR detection experiments, taking the Salmonella invA gene as an example, the detection limit after applying this method was determined as follows: beads were first added to the bacterial culture, followed by sonication, centrifugation, and the supernatant was collected for detection. The linear range was from 1.36 × 10⁻⁶.) 1 CFU / mL up to 1.36 × 10⁻⁶ 8 CFU / mL, R of the standard curve 2 The detection limit can reach 0.9946, demonstrating high performance. After diluting the bacterial solution and then adding beads for ultrasonication, the results show that for low-concentration bacterial solutions, the detection limit can reach as low as 6.8 CFU / mL, and the effect is significantly better than thermal lysis. Amplification of water in an ultrasonic water bath was not possible, demonstrating the anti-contamination characteristics of this method.

[0066] The amplification curve and standard curve of qPCR detection after ultrasonic grinding are shown in the figure. Figure 6 Figure A shows the amplification curves of qPCR amplification after serial dilution. Figure B shows the standard curve with a linearity of 0.9946.

[0067] The bacteria were first diluted with PBS before amplification. The positive rate results showed that the limit of detection (LOD) for thermal lysis at 95°C for 15 minutes was 10. 1 The detection limit for ultrasonic bead grinding is 6.8 CFU / mL, while the detection limit for ultrasonic bead grinding is 6.8 CFU / mL.

[0068] Since the method provided by this invention can detect bacteria as low as 6.8 CFU / mL, it holds promise for culture-free detection. The detection process involves food sample preparation (generally homogenization, though preparation methods vary depending on the food sample), which typically takes less than 10 minutes. Following this, sample pretreatment is performed, usually by filtration or centrifugation to obtain the supernatant, a process generally taking less than 5 minutes. Next, nucleic acid extraction is performed, taking 3 minutes. Finally, the gold standard qPCR method is used to detect the target pathogen, thus revealing the microbial contamination status of the food sample.

[0069] The feasibility of this nucleic acid extraction method was tested using low-concentration spiked experiments on seven different food samples. The food samples included chicken offal, milk, coconut juice, chicken legs, wastewater from a chicken processing counter, orange juice, and apple juice. As shown in Figure A, all samples could be spiked to 1.36 × 10⁻⁶ using a low-power water bath ultrasonic lysis method for foodborne pathogens. 1 CFU / mL. Figure B shows the concentration of 1.36 × 10⁻⁶ CFU / mL. 4After CFU / mL of bacteria, the final measured Ct value change shows that, apart from milk components having a significant impact on the test results, other food samples can be tested for Salmonella normally.

[0070] Actual sample testing using the method of this invention is shown in the figure. Figure 7 Figure A shows the positive rates of different samples under different bacterial addition conditions. Figure B shows the positive rates of different samples at the same bacterial concentration (10⁻⁶). 4 Ct values ​​of qPCR with added CFU / mL (milk 33.36, chicken offal 30.25, apple juice 29.67, coconut juice 29.09, sewage 28.11, orange juice 26.52, chicken leg 26.31).

[0071] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for lysing foodborne pathogens, characterized in that, include: ABS microspheres were added to a solution sample of foodborne pathogens, and the ABS microspheres were subjected to high-frequency vibration by ultrasound to achieve bacterial lysis. The particle size of the ABS microspheres was 1.0±0.2 mm. The ultrasound parameters were 0.12±0.01 W / mL and 40±3 kHz. The bacterial content in solution samples of foodborne pathogens is 6.8~13.6 CFU / mL, 13.6~13600 CFU / mL, or >13600 CFU / mL.

2. The method for lysing foodborne pathogens according to claim 1, characterized in that, The ultrasound treatment time was 2.5 ± 0.1 min.

3. The method for lysing foodborne pathogens according to claim 1, characterized in that, Add 0.2 ± 0.01 g of ABS beads to each 1 mL solution sample of foodborne pathogens.

4. The method for lysing foodborne pathogens according to any one of claims 1-3, characterized in that, Ultrasound was performed using a low-power water bath ultrasonic machine; the solution sample of foodborne pathogens was placed in a centrifuge tube; the angle between the centrifuge tube and the ultrasonic transducer during the ultrasonic process was 90±5°.

5. The method for lysing foodborne pathogens according to claim 1, characterized in that, The solution sample for foodborne pathogens is either a cultured or uncultured food sample to be tested.

6. The method for lysing foodborne pathogens according to claim 5, characterized in that, The food samples to be tested were from at least one of the following sources: chicken offal, milk, coconut milk, chicken legs, wastewater from the chicken counter, orange juice, and apple juice.

7. The method for lysing foodborne pathogens according to claim 1, characterized in that, Foodborne pathogens include Gram-positive and / or Gram-negative bacteria.

8. The method for lysing foodborne pathogens according to claim 7, characterized in that, Gram-positive bacteria include Bacillus cereus ( Bacillus cereus ); and / or, Gram-negative bacteria include Escherichia coli (Escherichia coli) ) or Salmonella typhimurium ( Salmonella Typhimurium ).

9. The application of the lysis method of foodborne pathogens according to any one of claims 1-8 in the detection of foodborne pathogens for non-disease diagnostic purposes.

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