Cracking method of food-borne pathogenic bacteria and application thereof

Abstract

The invention relates to the technical field of food safety, in particular to a low-power water bath ultrasonic cracking method for food-borne pathogenic bacteria and application thereof. According to the method, a low-power water bath ultrasonic machine is used, ABS small balls with the size of about 1 mm are added into a solution sample, high-frequency vibration of the ABS small balls is caused by ultrasonic waves, a local high sound pressure field and a high-density cavitation field generated by vibration can rapidly crack bacteria, meanwhile, the movement of the small balls can accelerate rapid substance exchange of solutions at different positions in a tube, and the bacteria can be rapidly decomposed. Therefore, 1 mL of bacterial sample to be detected can be split within 3 minutes. The method provided by the invention is high in cracking efficiency and short in consumed time. When the method is applied to detection of food-borne pathogenic bacteria, nucleic acid purification and enrichment are not required to be performed on the lysis solution, so that the time and labor cost required by nucleic acid detection can be greatly shortened, and a novel efficient, economical and practical bacterium lysis and nucleic acid extraction method is provided for the field of food safety detection.

Description

Technical Field

[0001] The invention relates to the technical field of food safety, and in particular to a method for lysing foodborne pathogenic bacteria using low-power water bath ultrasound and an application thereof. Background Art

[0002] In the field of food safety, rapid and accurate detection of foodborne pathogens is an important part of protecting public health. According to national standards such as GB 4789.4-2016, the sampling stage of foodborne pathogen detection often requires pre-enrichment and enrichment treatment to ensure that the number of bacteria in the sample reaches the detection limit of the detection method. This process takes at least 8 to 18 hours. However, traditional detection methods such as culture method and PCR gel electrophoresis method, although the test results are accurate, have problems such as long time consumption, complex operation, and high cost, which can hardly meet the needs of modern food safety detection for rapidity and efficiency.

[0003] In recent years, with the rapid development of molecular biology technology, qPCR (real-time fluorescence quantitative nucleic acid amplification detection technology) has been widely used in the detection of foodborne pathogens due to its advantages of high sensitivity, high specificity and rapidity. The qPCR method realizes the rapid quantitative detection of pathogens by directly detecting bacterial nucleic acids in samples. At present, the qPCR detection method has become the "gold standard" method for the detection of many pathogens. However, to perform qPCR detection, it is first necessary to extract high-quality and high-concentration nucleic acids from the sample, and bacterial lysis is a key step in nucleic acid extraction, and its efficiency and cost directly affect the performance of the entire detection process.

[0004] Traditional bacterial lysis methods include chemical lysis, thermal lysis, and ultrasound. Although chemical lysis is highly efficient, the experimental process is complex, requires additional nucleic acid purification steps, and has high reagent costs. It also requires professional laboratory conditions and professional experimental operators. These shortcomings make qPCR testing require additional laboratory costs, time costs, and labor costs; thermal lysis is relatively inefficient and prone to nucleic acid aerosol contamination; ultrasonic bacterial disruption relies on high-power ultrasonic processing equipment. There is currently a lack of related pathogen nucleic acid extraction methods, but it usually requires expensive ultrasonic lysis instruments and longer lysis times, which also increases the cost of testing.

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

[0006] In order to solve the above technical problems, the present invention provides a method for lysing foodborne pathogenic bacteria using low-power water bath ultrasound and its application.

[0007] Specifically, the technical solution of the present invention is as follows: In a first aspect, the present invention provides a method for lysing foodborne pathogenic bacteria, comprising: adding ABS beads to a solution sample of foodborne pathogenic bacteria, and using ultrasound to cause high-frequency vibration of the ABS beads, thereby achieving bacterial lysis; the particle size of the ABS beads is 1.0±0.2 mm; and the ultrasonic parameters are 0.12±0.01 W / mL, 40±3 KHz.

[0008] Preferably, the ultrasonic treatment time is 2.5±0.1 min.

[0009] Preferably, 0.2±0.01 g of ABS beads are added to every 1 mL of the solution sample of foodborne pathogens.

[0010] Preferably, the solution sample of foodborne pathogenic bacteria is placed in a centrifuge tube; during the ultrasonic process, the angle between the centrifuge tube and the ultrasonic transducer is 90±5°.

[0011] Preferably, the solution sample of foodborne pathogenic bacteria is a cultured or uncultured food sample to be tested.

[0012] Preferably, the source of the food sample to be tested is at least one of chicken viscera, milk, coconut milk, chicken legs, chicken counter sewage, orange juice, and apple juice.

[0013] Preferably, the bacterial content in the solution sample of foodborne pathogenic bacteria is less than 6.8 CFU / mL, 6.8-13.6 CFU / mL, 13.6-13600 CFU / mL or greater than 13600 CFU / mL.

[0014] Preferably, the foodborne pathogenic bacteria include Gram-positive bacteria and / or Gram-negative bacteria.

[0015] Preferably, the Gram-positive bacteria include Bacillus cereus ATCC 14579; and / or, the Gram-negative bacteria include Escherichia coli O157:H7 ATCC 43888 or Salmonella typhimurium ATCC 14028.

[0016] In a second aspect, the present invention provides an application of the foodborne pathogenic bacteria lysis method in the detection of foodborne pathogenic bacteria for non-disease diagnosis purposes.

[0017] Beneficial effects: The present invention provides a method for lysing foodborne pathogenic bacteria using low-power water bath ultrasound and its application. A low-power water bath ultrasound machine is used to add ABS beads of about 1 mm in size to a solution sample, and ultrasound is used to cause high-frequency vibration of the ABS beads. The local high sound pressure field and high-density cavitation field generated by the vibration will quickly lyse the bacteria. At the same time, the movement of the beads will accelerate the rapid material exchange of the solutions at different positions in the tube, so that 1 mL of the bacterial sample to be tested can be lysed within 3 minutes. The method provided by the present invention has high lysis efficiency and short time consumption. When applied to the detection of foodborne pathogenic bacteria, there is no need to purify the lysate for nucleic acid (no chemical reagents are involved), nor is there any need to enrich the lysate (high detection limit), which can greatly shorten the time and labor costs required for nucleic acid detection, and provide a new method of efficient, economical and practical bacterial lysis and nucleic acid extraction for the field of food safety detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be described below.

[0019] Figure 1 To verify the feasibility of the ultrasonic grinding method with beads, Figure A shows the schematic diagram of ultrasonic bead grinding with water bath ultrasound. The sample tube should be perpendicular to the transmission direction of the sound wave, which is conducive to the grinding beads absorbing the energy of the ultrasonic wave. Figure B shows 10 9 The difference in nucleic acid concentration between CFU / mL bacteria after ordinary ultrasound (without grinding beads) and ultrasonic bead grinding.

[0020] Figure 2 This is a comparison of the lysis performance between the ultrasonic bead grinding method and other methods. Figure A shows the difference between the ultrasonic bead grinding method and other physical bacterial lysis methods. Among them, there are significant differences between ultrasonic bead grinding and thermal lysis, repeated freezing and thawing, and ordinary ultrasound. Figure B shows the spectral data after ultrasonic bead grinding, thermal lysis, repeated freezing and thawing, and ordinary ultrasound. The characteristic peak of nucleic acid can be seen at 260 nm, confirming that this method can achieve the extraction of bacterial nucleic acid.

[0021] Figure 3 Figure 1 is a TEM characterization diagram of the ultrasonic bead grinding method. Figures A, B, C, and D show the TEM characterization diagrams of the original bacteria, ordinary ultrasound, thermal lysis, and ultrasonic bead grinding, respectively. It can be found that the bacterial morphology changes from complete to fragmented, indicating that ordinary ultrasound and thermal lysis can only partially lyse bacteria, but ultrasonic bead grinding can completely lyse bacteria. Figure E is a schematic diagram of the lysis effect of the above four methods. Figure F shows the size of the bacterial fragments calculated using the software, and the statistical data is consistent with the above results.

[0022] Figure 4This is a bead parameter optimization experiment. Figure A shows that 1 mm beads have the highest efficiency in nucleic acid extraction. Figure B shows that among different bead materials, although there is little difference between different materials, ABS plastic has the highest nucleic acid extraction efficiency and the lowest cost. Figure C shows that in terms of dosage, 0.2 g ABS beads have 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 shows that the sample tube is perpendicular to the ultrasonic transducer for the best effect.

[0023] Figure 5 This is an experiment to test the performance of ultrasonic grinding and lysing bacteria. Figure A shows the lysis efficiency of three types of bacteria, namely Bacillus cereus, with a lysis rate of 59.82%, Escherichia coli O157:H7, with a lysis rate of 66.53%, and Salmonella, with a lysis rate of 84.10%. Figure B shows the optimization of lysis time, and a lysis efficiency of more than 80% 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 the commercial kit.

[0024] Figure 6 The amplification curve and standard curve of qPCR detection after ultrasonic grinding, among which Figure A is the amplification curve of qPCR amplification after gradient dilution. Figure B is the standard curve, and the linearity is 0.9946.

[0025] Figure 7 This is an actual sample test using the method of the present invention, wherein Figure A shows the positive rates of different samples under different bacterial addition conditions, and Figure B shows the Ct values ​​of qPCR of different samples under the same bacterial concentration.

[0026] Figure 8 The following are the results of finite element simulation analysis, where Figure A is the force analysis of a single bead in water bath ultrasound. Figure B is the interaction of multiple beads in water bath ultrasound. Figure C is the sound velocity and sound pressure distribution from single to multiple beads. Figure D is the total sound pressure distribution inside beads of different sizes. Figures E, F, G, and H are the sound velocity field and flow velocity field when the beads are not vibrating and the sound velocity field and flow velocity field when the beads are vibrating. DETAILED DESCRIPTION

[0027] The present invention proposes a low-power water bath ultrasonic foodborne pathogenic bacteria lysis method, which aims to solve the problems of high cost and low efficiency of existing bacterial lysis methods. The method adds small balls to the sample solution and uses a low-power water bath ultrasonic cleaning machine commonly used in the laboratory for ultrasonic treatment, thereby achieving rapid and effective lysis of rod-shaped Gram-positive bacteria and Gram-negative bacteria. This method not only simplifies the experimental process and avoids expensive equipment and reagent costs, but also significantly shortens the time for bacterial lysis and nucleic acid extraction, thereby improving detection efficiency.

[0028] Specifically, the low-power water bath ultrasonic bacterial lysis method provided by the present invention can complete the lysis of bacteria in 1 mL of sample solution within 3 minutes. The detection limit of qPCR performed using this method can reach 13.6 CFU / mL, which is much higher than the detection sensitivity of traditional methods. Since no lysis solution is added, this method does not require a nucleic acid purification step, and the higher detection limit also means that this method does not require nucleic acid enrichment, greatly shortening the time and labor costs required for nucleic acid detection. This method can be completed using only a commonly used low-power (0.12 W / mL, 40±3KHz) water bath ultrasonic cleaning machine (the price is less than one-tenth of the ultrasonic cell disruptor used in the laboratory) and a 1mm ABS ball, which greatly reduces the cost of bacterial lysis and nucleic acid extraction, and provides a new method of efficient, economical and practical bacterial lysis and nucleic acid extraction for the field of food safety testing.

[0029] The following is a specific description in conjunction with the research process of the present invention.

[0030] The present invention proposes a novel bacterial lysis method, using a low-power water bath ultrasonic machine, adding 1mm ABS beads to the solution sample, and ultrasound causes the ABS beads to vibrate at high frequencies. The local high sound pressure field and high-density cavitation field generated by the vibration will quickly lyse the bacteria, and at the same time, the movement of the beads will accelerate the rapid material exchange of the solutions at different positions in the tube, so that 1mL of the tested bacterial sample can be lysed within 3 minutes. During the research process, the present invention optimizes the size, material, dosage, centrifuge tube placement, ultrasonic time, and types of effective bacteria of the ultrasonic beads in the tube; and compares the lysis efficiency of different lysis methods to prove the effectiveness of the method. At the same time, the present invention uses Salmonella as an example, and through qPCR, verifies the effectiveness of the method for foodborne pathogen detection, determines the minimum detection limit of the method, and proves the feasibility of the method in the field of foodborne pathogen detection through real samples.

[0031] First, the present invention combines low-power water bath ultrasound with ABS beads to achieve lysis. First, a low-power water bath ultrasound machine is used under specific conditions (ultrasonic power 0.12 W / mL, frequency 40±3KHz) in combination with 1 mm ABS beads (the beads are made of acrylonitrile-butadiene-styrene copolymer, with a diameter of 1 mm, and the amount in a 1.5 mL centrifuge tube commonly used 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 are used to achieve rapid bacterial lysis.

[0032] Secondly, the present invention optimizes the ultrasonic ball in the tube, specifically including: optimizing the size (diameter is about 1 mm), material (acrylonitrile-butadiene-styrene copolymer), and dosage (the dosage in a 1.5 mL centrifuge tube commonly used in the laboratory is 0.2±0.01 g) of the ultrasonic ball in the tube. These optimizations play a key role in improving the lysis efficiency and ensuring the lysis effect, and ensure the realization of the technical effect of the present invention.

[0033] Furthermore, the present invention optimizes and controls the operating procedures and conditions. First, the placement of the centrifuge tube and the ultrasonic time are optimized: the placement of the centrifuge tube in the ultrasonic equipment (the centrifuge tube should be perpendicular to the ultrasonic transducer) and the ultrasonic treatment time (2.5±0.1min) have a significant effect on the lysis effect of the present invention. The lysis process is also standardized: sample preparation (in accordance with national standard GB 4789.4-2016), taking 1mL of sample, adding 0.2±0.01g of 1mm diameter ABS beads, ultrasonic treatment for 3 minutes, and then aspirating the supernatant sample solution for qPCR detection. The entire lysis process is preferably implemented according to standardized operating steps to ensure the repeatability and consistency of the technology.

[0034] In addition, the present invention also verifies the application effect of the scheme by data. First, the lysis efficiency is compared and verified: by comparing the lysis efficiency data of different lysis methods (such as chemical lysis, thermal lysis, traditional ultrasonic method, etc.), it is confirmed that the scheme provided by the present invention has obvious superiority in these comparisons. The minimum detection limit is verified by qPCR: the qPCR technology is used to verify the effectiveness of this method in the detection of foodborne pathogens (such as Salmonella). For the specific qPCR experimental design, operation steps, result analysis and measurement data of the minimum detection limit (13.6 CFU / mL), please refer to the contents recorded in the embodiment. In view of the fact that the present invention claims to protect the application of the foodborne pathogen lysis method in the detection of foodborne pathogens for non-disease diagnosis purposes, the qPCR related methods disclosed in the embodiments or the undisclosed related contents of foodborne pathogen detection using the foodborne pathogen lysis method also fall within the protection scope of the present invention. The present invention also verifies the feasibility of the application of real samples: the feasibility of the method in the field of foodborne pathogenic bacteria detection is verified by real food samples. Based on the experimental data and analysis results, it is proved that the method of the present invention is reliable and practical in practical applications.

[0035] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] 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 each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0037] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "specific implementations", or "some specific implementations" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the embodiment of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0038] In the examples provided in this specification, if no specific techniques or conditions are specified, the techniques or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0039] 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 the Applied Biosystems QuantStudio 3qPCR instrument from Thermo Fisher Scientific. Nucleic acid concentrations were determined using the nanoDrop one C ultra-micro UV-visible spectrophotometer from Thermo Fisher Scientific. Homogenizing bags and homogenizing mixers BagMixer 400CC were purchased from Interscience. Primers for qPCR were synthesized by Shanghai Shenggong Biotechnology Co., Ltd. The qPCR master mix used was Taq ProUniversal SYBR qPCR Master Mix, from Nanjing Novi Biotechnology Co., Ltd. The commercial kit used in the control experiment was the Magnetic Universal Genomic DNA Kit purchased from Tiangen Biochemical Technology Co., Ltd. The temperature sensor was a TM-902C K-type thermocouple purchased from Taizhou Novi Electrical Equipment Co., Ltd. There were no special requirements for other materials and instruments.

[0040] In the following examples, the bacterial strains used 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 cultured at 37°C for 16 hours to ensure optimal growth (this culture method allows the bacteria to proliferate to a concentration suitable for further experiments).

[0041] In the following examples, DNA concentration is expressed as mean ± standard deviation (SD). Each experiment was repeated at least five times. A t-test was used for significance analysis. A P value of less than 0.05 was accepted as statistically significant. Multiphysics simulation was performed using COMSOL 6.2 software. The 3D printing modeling software was Solidworks 2024. The Ct value was calculated by the threshold method, that is, 10 standard deviations were added to the mean of the signal values ​​of the first ten cycles calculated by Design&Analysis Software 2.7.0. ImageJ software (used to analyze the area of ​​transmission electron microscopy (TEM) characterization photos. MATLAB R2024a and MATLAB cftool were used to perform linear regression analysis. Student's t-test was used for significance analysis using GraphPad software.

[0042] In the following examples, the supernatant of the bacterial extract was used for nucleic acid concentration measurement in subsequent proof-of-concept ultrasonic grinding experiments. 0.25 g of 1 mm diameter ABS beads were added to a 1.5 mL centrifuge tube and then mixed with 1 mL of the 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 min of ultrasonic bead grinding lysis, the supernatant was aspirated and centrifuged at 12,000 rpm to remove impurities or directly processed for 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, because 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.

[0043] In the following examples, ultrasonic bead grinding was used to replace the traditional nucleic acid extraction step in the kit. 8 The bacterial solution of CFU / mL was dissolved in ultrapure water and then lysed. The supernatant after lysis was serially diluted and detected by qPCR. After ultrasonic bead grinding, serial dilution was performed. qPCR amplification and signal reading were performed using nucleic acids extracted at different concentrations. In order to compare with the thermal lysis method, 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. 20 μL of PCR amplification 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. A melting curve experiment was performed after the qPCR detection experiment to exclude the interference of nonspecific amplification.

[0044] In the following examples, bacterial counts were determined using the LB agar plate spread method. After incubation at 37°C for 12 hours, colonies were counted to assess the bacterial load in the sample. First, a Salmonella solution (1.36 × 10 8 CFU / mL) were added to the food samples. The standard method GB 4789.4-2016 was strictly followed to ensure the integrity and reliability of the samples used in this study. In brief, 25 mL of food samples were first mixed with 225 mL of sterile phosphate buffered saline (PBS) and homogenized for 2 min. Then, 0.25 g of ABS beads were mixed with 1 mL of sample solution, ultrasonically ground for 3 min, and graded diluted to 1.36 × 10 2 to 1.36 × 105 The concentration of CFU / mL was measured. Finally, the qPCR method was used to detect bacterial nucleic acids. Chicken thighs purchased from supermarkets, sewage collected at chicken counters, and chicken viscera were selected as poultry samples. Milk, coconut juice, orange juice, and apple juice on the shelves were also purchased as beverage samples.

[0045] Example 1 This embodiment provides a method for lysing foodborne pathogenic bacteria. 1 mL of a solution sample of foodborne pathogenic bacteria (in accordance with the national standard GB 4789.4-2016) is added to a 1.5 mL centrifuge tube, and then 0.2 g of ABS beads with a particle size of 1 mm are added. A low-power water bath ultrasonic machine is used, and the ultrasonic parameters are set to 0.12 W / mL and the frequency is 40±3 KHz, and the ultrasonic treatment is performed for 2.5 minutes.

[0046] Example 2 This embodiment describes the experimental contents of the research related to the scheme of Embodiment 1 in conjunction with the accompanying drawings.

[0047] The main principle of bacterial lysis is to use the beads to repeatedly expand and contract under the interaction of the high-pressure area and the low-pressure area of ​​ultrasound. After the beads shrink, the bacteria and the surrounding solution are pressurized. After the beads expand, the pressure of the bacteria and the surrounding solution decreases. At the same time, with the cavitation effect, after such a cycle, the bacteria can be quickly lysed and the DNA is released. Ultimately, this method can improve the sensitivity of qPCR detection.

[0048] During grinding, the beads are added to the centrifuge tube. Under the stimulation of ultrasound, the beads vibrate at high frequency, and the acoustic flow, cavitation and convection effects induced by them jointly complete the bacterial lysis and nucleic acid extraction in large volume (1 mL) samples. Among them, the acoustic flow synergistic cavitation effect can achieve bacterial lysis, while the acoustic flow convection effect can achieve rapid material exchange, thereby improving the lysis efficiency.

[0049] The process of ultrasonic bead grinding for foodborne pathogen detection includes three steps, namely the first step of food sample material and pretreatment, including homogenization, filtration, centrifugation, pre-enrichment steps, etc. The specific operation of this step needs to refer to the national standard GB 4789.1-2016 according to actual needs. The second step is bacterial lysis and nucleic acid extraction. Add an appropriate amount of beads to a centrifuge tube, place it vertically on an ultrasonic cleaner, and sonicate for 3 minutes. The third step is to mix the sample with the qPCR reaction system and then perform qPCR detection. This method greatly improves the bacterial lysis efficiency of low-power ultrasound, reduces the detection cost, and simplifies the detection steps of foodborne pathogens.

[0050] The feasibility of ultrasonic grinding with beads was verified in Figure 1, where Figure A shows a schematic diagram of ultrasonic grinding in a water bath. The sample tube should be perpendicular to the direction of sound wave transmission, which is conducive to the grinding beads absorbing the energy of the ultrasonic wave (the sample tube and the ultrasonic transducer should be at 90° to ensure the best and fastest lysis). Figure B shows 10 9 The difference in nucleic acid concentration between CFU / mL bacteria after ordinary ultrasound (without grinding beads) and ultrasonic bead grinding.

[0051] The comparison of the pyrolysis performance between the method of the present invention and other methods is shown in Figure 2 , among which, Figure A shows the difference between ultrasonic bead grinding and other physical bacterial lysis methods. Among them, there are significant differences between ultrasonic bead grinding and thermal lysis, repeated freezing and thawing, and ordinary ultrasound (after adding beads, the lysis efficiency is significantly higher than thermal lysis, and significantly higher than the ultrasound without beads at the same ultrasound time and ultrasound placement). Figure B shows the spectral data after ultrasonic bead grinding, thermal lysis, repeated freezing and thawing, and ordinary ultrasound. The characteristic peak of nucleic acid can be seen at 260 nm, confirming that this method can achieve the extraction of bacterial nucleic acid (using nano drop to measure the absorption peak of the extracted nucleic acid, it can be seen that there is an obvious absorption peak at 260 nm, which proves the feasibility of extracting nucleic acid and the integrity of the nucleic acid chain).

[0052] TEM characterization pictures Figure 3 , among which, Figure A, Figure B, Figure C and Figure D respectively show the TEM characterization images of the four methods from the original bacteria, ordinary ultrasound, thermal lysis, and ultrasonic bead grinding. It can be found that the bacterial morphology changes from complete to fragmented, indicating that ordinary ultrasound and thermal lysis can only partially lyse bacteria, but ultrasonic bead grinding can completely lyse bacteria. Figure E is a schematic diagram of the lysis effect of the above four methods. Figure F shows the size of the bacterial fragments calculated by the software, and the statistical data is consistent with the above results.

[0053] In the low-power water bath ultrasonic bead milling method, the force and movement of the beads in water bath ultrasound are analyzed. The beads in the solution are mainly affected by acoustic radiation force, buoyancy, gravity, fluid resistance and the interaction force between the beads. Among them, the force on a single bead is analyzed. When a single bead is in ultrasound, the bead is affected by gravity, buoyancy, fluid resistance and acoustophoretic radiation force, and cavitation bubbles are generated on the surface due to friction. The force when there are multiple beads is analyzed. In addition to the above forces, they are also affected by the interaction force between the beads, which converts ultrasonic energy into heat, which is expected to promote the occurrence of cavitation. Multiple beads will produce more cavitation bubbles.

[0054] By analyzing the physical fields related to ultrasonic grinding through finite element simulation, it was found that the aggregation of multiple beads can significantly increase the local sound pressure. At the same time, the magnitude of the local sound pressure will mainly depend on the size of the beads. The appropriate bead size and number of beads can provide a large attachment area for cavitation, thereby significantly enhancing the acoustic cavitation effect. At the same time, the enhancement of local sound velocity and flow velocity generated by the high-frequency vibration of the beads can also help improve the efficiency of nucleic acid extraction. Figure 8 .

[0055] In order to verify that the interaction between multiple beads will increase the local acoustic pressure, the sound velocity and sound pressure distribution of 1 mm diameter beads in a single, two, and four groups in a contracted state were analyzed using two-dimensional finite element analysis, and the sound pressure distribution at the center of four beads of different bead sizes was explored. The simulation results show that the sound pressure inside the 1.5 mm diameter beads is the highest when four beads are gathered.

[0056] By comparing the acoustic convection effects (sound velocity distribution and flow velocity distribution) of the beads when they are vibrating and when they are not vibrating, the verification and simulation results show that: the high gradient sound velocity is concentrated between the beads; when the beads vibrate at high frequency, they will drive the high-speed movement of the fluid, thereby achieving efficient material exchange, thereby improving the grinding efficiency.

[0057] Bead parameter optimization experiment see Figure 4 , among which, Figure A shows that 1 mm beads have the highest efficiency in nucleic acid extraction. Figure B shows that among different bead materials, although there is little difference between different materials, ABS plastic has the highest nucleic acid extraction efficiency and the lowest cost. Therefore, ABS plastic was selected as the material for grinding beads in subsequent experiments. Figure C shows that in terms of dosage, 0.2 g ABS beads have 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 is perpendicular to the ultrasonic transducer for the best effect. The above experimental content optimizes and verifies the placement of the centrifuge tube (the best angle with the ultrasonic transducer is 90°), the material of the ultrasonic ball in the centrifuge tube (ABS ball has the best effect), the diameter of the ultrasonic ball in the centrifuge tube (1 mm is the best), and the amount of ultrasonic ball in the 1.5 mL centrifuge tube (0.2±0.01 g is the best).

[0058] In the low-power water bath ultrasonic bead milling method, in addition to the movement of the beads, the bacteria are also subjected to non-uniform shear force between the beads. Specifically, the rod-shaped bacteria are subjected to non-uniform acoustic radiation force (compression and expansion force of the acoustic pressure wave). At the same time, when the bacteria are close to the beads, they are also subjected to shear force and rupture impact force (cavitation bubble impact) caused by cavitation bubbles. Other forces such as fluid shear force (compression force) and disintegration caused by denaturation of bacterial proteins can also promote bacterial lysis. In the ultrasonic field, the surface acceleration of the beads is tangential to the surface, which promotes the friction between the bead surface and the solution and promotes the cavitation effect. The forces on the bacteria are analyzed. Like the beads, the forces on the bacteria are mainly gravity, buoyancy, fluid resistance, acoustophoretic radiation force and pressure of the beads. By analyzing the surface velocity direction of the grinding beads under the ultrasonic field, it is shown that high surface sound velocity will bring more cavitation effect and help bacterial lysis. The sound velocity on the bacterial surface between the two beads was simulated, and the non-uniform sound velocity on the bacterial surface can cause the bacteria to break faster.

[0059] Three different bacteria, Bacillus cereus ATCC 14579 (Gram-positive bacteria), Escherichia coli O157:H7 ATCC 43888 (Gram-negative bacteria), and Salmonella typhimurium ATCC 14028 (Gram-negative bacteria), were selected to test the lysis efficiency of the lysis method of the present invention, the commercial kit, 70°C water bath heating, and lysis solutions such as proteinase K and lysozyme.

[0060] The results of the cracking efficiency test are shown in Figure 5 , where Figure A shows the lysis efficiency of three bacteria, namely Bacillus cereus, with a lysis rate of 59.82%, Escherichia coli O157:H7, with a lysis rate of 66.53%, and Salmonella, with a lysis rate of 84.10% (the present invention has a high lysis efficiency for these three common foodborne pathogens). Figure B shows the optimization of the lysis time, and a lysis efficiency of more than 80% can be achieved in 150 seconds (time optimization: 150 s can complete 1 mL 10 9 Figure C compares the lysis efficiency of different lysis methods. The lysis efficiency of ultrasonic bead grinding is second only to that of the commercial kit (ultrasound + beads for 1 mL 10 9 The lysis efficiency of CFU / mL Salmonella liquid is comparable to that of the commercial kit. However, ultrasound + beads only takes 3 minutes, while the commercial kit takes more than 15 minutes).

[0061] The qPCR melting curves of the three bacteria were measured after lysis. The results showed that the correct sequences could be amplified respectively, which verified the feasibility of qPCR detection after ultrasound (the qPCR detection experiment was carried out by the method of the present invention. The invA gene of Salmonella was taken as an example to determine the detection limit after the use of the method: beads were first added to the bacterial solution, ultrasound was performed, and the supernatant was centrifuged after ultrasound for detection. The linear range was from 1.36×10 1 CFU / mL to 1.36×10 8 CFU / mL, R of standard curve 2 The detection limit can reach 0.9946, which has a high detection performance. After diluting the bacterial solution, the beads were added for ultrasonication. The results show that for low-concentration bacterial solution, the detection limit can reach 6.8 CFU / mL, and the effect is significantly better than thermal lysis. The water in the ultrasonic water bath was amplified, and the results showed that amplification could not be performed, which demonstrated the anti-pollution characteristics of this method.

[0062] The amplification curve and standard curve of qPCR detection after ultrasonic grinding are shown in Figure 6 , where A is the amplification curve of qPCR amplification after gradient dilution. B is the standard curve with a linearity of 0.9946.

[0063] The bacteria were diluted with PBS before amplification. The positive rate test results showed that the minimum detection limit of thermal lysis at 95 degrees Celsius for 15 minutes was 10 1 CFU / mL, while the lowest detection limit of ultrasonic bead milling was 6.8 CFU / mL.

[0064] Since the method provided by the present invention can detect bacteria as low as 6.8 CFU / mL, it is expected to achieve culture-free detection. The detection process is food sample preparation (generally homogenization, different food samples have different preparation methods), this process usually takes less than 10 minutes, followed by sample pretreatment, generally filtering or centrifuging to obtain the supernatant, this process usually takes less than 5 minutes. Next, the nucleic acid extraction step is performed, which takes 3 minutes. The last step is to use the "gold standard" qPCR method to detect the target pathogen, and the microbial contamination of the food sample can be obtained.

[0065] Seven different food samples were used for low-concentration sample spike experiments to test the feasibility of the nucleic acid extraction method. The food samples were chicken offal, milk, coconut milk, chicken legs, chicken counter sewage, orange juice, and apple juice. As shown in Figure A, all samples could be tested to 1.36×10 1 CFU / mL. Figure B shows the spiked 1.36×10 4After the CFU / mL bacteria were detected, the final measured Ct value changed. It can be seen that except for the milk component which has a greater impact on the test results, other food samples can be tested for Salmonella normally.

[0066] The actual sample test using the method of the present invention is shown in Figure 7 , where A shows the positive rate of different samples under different bacterial addition conditions. B shows the positive rate of different samples under the same bacterial concentration (10 4 CFU / mL) were added (milk was 33.36, chicken viscera was 30.25, apple juice was 29.67, coconut milk was 29.09, sewage was 28.11, orange juice was 26.52, and chicken legs were 26.31).

[0067] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for lysing foodborne pathogenic bacteria, characterized in that: include: ABS beads were added to the solution sample of foodborne pathogens, and ultrasound was used to cause high-frequency vibration of the ABS beads, thereby achieving bacterial lysis; the particle size of the ABS beads was 1.0±0.2mm; the ultrasound parameters were 0.12±0.01 W / mL, 40±3 kHz.

2. The method for lysing foodborne pathogenic bacteria according to claim 1, characterized in that: The ultrasonic treatment time was 2.5±0.1min.

3. The method for lysing foodborne pathogenic bacteria according to claim 1 or 2, characterized in that: Add 0.2±0.01 g of ABS beads to every 1 mL of solution sample of foodborne pathogens.

4. The method for lysing foodborne pathogenic bacteria according to any one of claims 1 to 3, characterized in that: Use a low-power water bath ultrasonic machine for ultrasonication; place a solution sample of foodborne pathogens in a centrifuge tube; during the ultrasonication process, the angle between the centrifuge tube and the ultrasonic transducer is 90±5°.

5. The method for lysing foodborne pathogenic bacteria according to any one of claims 1 to 4, characterized in that: The solution sample of foodborne pathogens is a cultured or uncultured food sample to be tested.

6. The method for lysing foodborne pathogenic bacteria according to claim 5, characterized in that: The source of the food sample to be tested is at least one of chicken viscera, milk, coconut milk, chicken legs, chicken counter sewage, orange juice, and apple juice.

7. The method for lysing foodborne pathogenic bacteria according to any one of claims 1 to 6, characterized in that: 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.

8. The method for lysing foodborne pathogenic bacteria according to any one of claims 1 to 7, characterized in that: Foodborne pathogens include Gram-positive and / or Gram-negative bacteria.

9. The method for lysing foodborne pathogenic bacteria according to claim 8, characterized in that: Gram-positive bacteria include Bacillus cereus; and / or, Gram-negative bacteria include Escherichia coli or Salmonella typhimurium.

10. Use of the method for lysing foodborne pathogenic bacteria according to any one of claims 1 to 9 in detecting foodborne pathogenic bacteria for non-disease diagnosis purposes.

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