Rapid and accurate detection method for pathogenic bacteria

Abstract

The invention discloses a rapid and accurate detection method for pathogenic bacteria. The method comprises the following steps: immobilizing bacteriophages on the surfaces of magnetic nanoparticles through physical adsorption or chemical reaction, selectively enriching pathogenic bacteria in a complex sample by adopting the bacteriophage functionalized magnetic nanoparticles, quickly cracking the pathogenic bacteria on the surfaces of the magnetic nanoparticles by using CTAB (cetyltrimethyl ammonium bromide), releasing ATP in the bacteria, and quantitatively analyzing ATP by using bioluminescence. Further calculating the content of the pathogenic bacteria; the detection method is free of enrichment and amplification, can complete capture, enrichment and detection of pathogenic bacteria within 20 minutes, and can specifically detect viable bacteria of target bacteria in various complex matrix (such as food) samples due to the fact that bacteriophages are adopted as recognition molecules.

Description

Technical Field

[0001] The invention belongs to the technical field of food safety detection, and specifically relates to a rapid detection method for pathogenic bacteria. Background Art

[0002] Pathogenic bacteria (such as Salmonella, Staphylococcus aureus, etc.) can cause food poisoning or disease, seriously endangering human health. According to statistics from the World Health Organization, about 115 million people are infected with Salmonella every year. Although most people will recover on their own in a short period of time, about 370,000 people die from it. Therefore, the detection of pathogenic bacteria in food is of great significance to protecting people's health.

[0003] At present, the traditional colony counting method is still used as a "gold standard" for detecting Salmonella in food, but its detection cycle is long, usually taking 3-4 days. In recent years, in order to meet the needs of rapid and accurate detection of Salmonella, a variety of different strategies have been developed based on immunology, molecular biology and other methods. Immunology-based methods, such as enzyme-linked immunosorbent assay (ELISA), are easy to operate and have a short detection time, but require antibodies as recognition elements. Antibodies usually have a long preparation cycle and are relatively expensive, and cannot distinguish between live and dead bacteria. Methods such as polymerase chain reaction (PCR) based on molecular biology are fast, highly specific, and highly sensitive, but this technology requires professional technicians to operate; at the same time, in order to reduce the influence of complex matrices in food, cumbersome pre-treatment steps are required; because it is based on DNA detection, it is also unable to distinguish between live and dead bacteria. Flow cytometry can distinguish between live and dead bacteria, but its instruments are expensive and require more complex calibration and operation.

[0004] ATP is a direct energy source that drives cell life activities and its content is stable in most living cells. Therefore, releasing intracellular ATP and quantifying it using luciferase is a method for detecting the total number of living cells. This method is simple to operate, takes a short detection time, and can distinguish between live and dead bacteria. However, since ATP is a ubiquitous bioenergy substance, this method cannot exclude the influence of non-target bacteria and complex sample matrices. For example, the direct detection limit of pathogenic bacteria in milk is 10 3 CFU / mL or so.

[0005] Bacteriophages are viruses that infect bacteria. They are host-specific and only recognize live host bacteria. In food safety and microbial testing, the bacterial capture efficiency usually needs to reach more than 50% ("Research Progress of Immunomagnetic Separation Technology in Rapid Detection of Foodborne Pathogens". Food Science, 2019, 40, 338-345.) In addition, the complex food matrix usually requires effective sample pretreatment to obtain accurate and reliable test results. This pretreatment step also prolongs the detection time and increases the detection cost. Summary of the invention

[0006] In response to the above problems, the present application provides a method for rapid and accurate detection of pathogenic bacteria. Without the need for bacterial enrichment or amplification, the capture, enrichment and detection of live pathogenic bacteria in complex samples can be completed within 20 minutes, thereby improving the accuracy of pathogenic bacteria detection and shortening the detection time and steps.

[0007] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0008] A rapid and accurate detection method for live pathogenic bacteria for non-medical purposes, comprising three steps: preparation of bacteriophage-functionalized magnetic nanoparticles, and magnetic separation and enrichment of samples and target bacteria.

[0009] 1) Take 5 mg of core-shell magnetic nanoparticles and place them in a 2 mL centrifuge tube. Incubate them with alcohol three times, each time for 3 min; then incubate them with sterile MES three times, each time for 5 min; then use a magnet to adsorb the magnetic nanoparticles to the tube wall, and remove the supernatant; then add 1 mL of the modifier to the centrifuge tube, ultrasonicate at room temperature for 30 min, and shake at 600 rpm for 1 h; again use a magnet to adsorb the magnetic nanoparticles to the tube wall, and remove the supernatant; then wash them with an equal volume of sterile PBS for 3 times, add 1 mL of 10 8 -10 12 PFU / mL phage, 180rpm, incubate at 37°C for 12h; the above phage is the phage corresponding to the target bacteria;

[0010] The centrifuge tube was washed three times with sterile PBS, 500 μL of 5.0% BSA (bovine serum albumin) solution was added, and the mixture was reacted at 180 rpm for 30 min. The magnetic nanoparticles were adsorbed on the tube wall using a magnet, and the BSA solution was discarded. The centrifuge tube was then washed three times with 1 mL of PBS, and finally resuspended with 1 mL of PBS to obtain functionalized magnetic nanoparticles.

[0011] 2) taking 1 ml of the sample and incubating it with 60-100 μL of the functionalized magnetic nanoparticles obtained in step 1) for 15 min (preferably at 37° C.);

[0012] 3) The supernatant was discarded, and 20 μL of 0.05% CTAB solution was added and incubated for 3 minutes; finally, the fluorescence intensity of ATP was detected using a commercially available enhanced ATP detection kit, and the live bacterial content of the target bacteria in the sample was obtained by substituting it into the regression equation.

[0013] The above samples are liquid samples or solid samples; liquid samples can be directly tested. If the sample is a solid sample, first take 2g of the sample and crush it to a particle size of less than 5mm, then add 18ml of sterile water to mix it to prepare a mixed solution, and then test the mixed solution. The above samples include but are not limited to environmental matrices such as water bodies, or food matrices such as milk, chicken, and duck.

[0014] The above-mentioned target bacteria include Salmonella, Listeria, Staphylococcus aureus, etc.; when the target bacteria is Salmonella, its regression equation is y=0.00992x+52.32; when the target bacteria is Listeria, its regression equation is y=0.0093x+59.78; when the target bacteria is Staphylococcus aureus, y=0.0105x+48.69. The above y and x are RLU (fluorescence intensity) and bacterial concentration, respectively.

[0015] The method of the present application first fixes bacteriophages on the surface of magnetic nanoparticles by physical adsorption or chemical reaction to obtain bacteriophage-functionalized magnetic nanoparticles; then uses the nanoparticles to selectively enrich specific pathogenic bacteria in complex samples; finally uses CTAB to quickly lyse the pathogenic bacteria on the surface of the magnetic nanoparticles to release adenosine triphosphate (ATP) in the bacteria; uses bioluminescence to quantitatively analyze ATP to further determine the content of the pathogenic bacteria; has the advantages of easy operation, strong specificity and high separation efficiency, and can quickly separate and enrich target pathogens from complex food samples; finally uses the ATP bioluminescence detection method to quickly and accurately detect pathogenic bacteria in complex samples in real time.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] 1. The detection method provided in this application does not require bacterial enrichment or amplification, and can complete the capture, enrichment and detection of pathogenic bacteria within 20 minutes.

[0018] 2. This application uses bacteriophage as a recognition molecule, which can identify live bacteria of specific bacteria. Compared with specific recognition elements such as antibodies and aptamers, the time for separation and purification of bacteriophage is shorter and the cost is the lowest, which is convenient for promotion and application.

[0019] 3. This application uses magnetic separation and enrichment for sample pretreatment, which can detect target bacteria in complex sample food, environment, and clinical samples, and achieve specific enrichment of target strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The zeta potential characterization results of Phage-MNPs;

[0021] Figure 2 It is the specific detection result of Phage-MNPs;

[0022] Figure 3 is the linear relationship between ATP bioluminescence signal RLU and Salmonella concentration;

[0023] Figure 4 Schematic diagram of the detection method of the embodiment. DETAILED DESCRIPTION

[0024] Unless otherwise specified, the raw materials and reagents involved in the examples are conventional commercially available products.

[0025] CTAB (hexadecyltrimethylammonium bromide), EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide), NHS (sodium salt of N-hydroxysulfosuccinimide), MES (2-morpholineethanesulfonic acid), and BSA (bovine serum albumin) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.;

[0026] The core-shell magnetic nanoparticles used in the embodiment are prepared according to the method disclosed in the document "Preparation and Characterization of Core-Shell Structured Fe3O4@SiO2 Nanoparticles [J]. Journal of Chengdu University of Technology (Natural Science Edition) 2019, 46(01): 118-122", and the particle diameter is about 500 nm.

[0027] PBS (phosphate buffered saline) was purchased from Sangon Biotechnology (Shanghai) Co., Ltd.

[0028] Modifier: EDC with a final concentration of 200 mg / mL and NHS with a final concentration of 100 mg / mL were added to a 25 mM MES (pH=6) solution, and the solution was obtained after sterilization by filtration using a sterile filter membrane with a pore size of 0.22 μm.

[0029] Salmonella phage PA13076, Listeria phage LipG2-5, and Staphylococcus aureus phage JS25 are all preserved by Jiangsu Academy of Agricultural Sciences. They are all conventional phages, as disclosed in the literature "Microchimica Acta, 2024, 191: 550", "Microbiological Research 2015, 172: 1–6)", and "Virus Genes, 2015, 50: 111–117)".

[0030] The host strains of Salmonella ATCC 13076, ATCC 25922, ATCC 36150, ATCC 29213, CMCC 26001, ATCC 35666, ATCC 29544, and Listeria monocytogenes (Lm007) were all standard strains purchased from the Chinese Culture Resource Bank.

[0031] Example 1 Preparation and Characterization of Phage-functionalized Magnetic Nanoparticles (Phage-MNPs)

[0032] 1) Take 5 mg of core-shell magnetic nanoparticles and place them in a 2 mL centrifuge tube. Incubate (sterilize) them three times with 75% (volume concentration) alcohol, each time for 3 min; then incubate (wash) them three times with sterile MES, each time for 5 min. Use a magnet to adsorb the magnetic nanoparticles to the tube wall, and remove the supernatant.

[0033] 2) Add 1 mL of the modifier to the centrifuge tube, ultrasonicate at room temperature for 30 min, and shake at 600 rpm for 1 h. Use a magnet to adsorb the magnetic nanoparticles to the tube wall again, remove the supernatant, and then wash with an equal volume of sterile PBS for 3 times, add 1 mL of 10 12 PFU / mL Salmonella phage PA13076, rotation speed 180rpm, incubation at 37℃ for 12h.

[0034] 3) Add 500 μL of 5.0% BSA (bovine serum albumin) solution, react at 180 rpm for 30 min, use a magnet to adsorb the magnetic nanoparticles on the tube wall, and discard the BSA solution. Wash the centrifuge tube three times with 1 mL of PBS, and finally resuspend with 1 mL of PBS to obtain Phage-MNPs, which are stored at 4°C for later use.

[0035] Both the carboxyl group -COOH and the bacteriophage have negative charges. Figure 1 The Zeta potential detection results of the prepared Phage-MNPs and the comparison Fe3O4@SiO2-NH2 and Fe3O4@SiO2-COOH (the two comparison materials are conventional materials, and this embodiment is prepared according to the method disclosed in the document "Journal of Chengdu University of Technology (Natural Science Edition), 2019, 46(01): 118-122.") (the detection method refers to the document "Microchimica Acta, 2024, 191: 550") indicate that the bacteriophage has been successfully modified on the surface of the material.

[0036] Example 2 Specificity of Phage-MNPs

[0037] 60 μL of Phage-MNPs solution obtained in Example 1 was mixed with 100 μL of 10 5CFU / mL of Salmonella host bacteria ATCC 13076, six interfering strains (ATCC 25922, ATCC 36150, ATCC 29213, CMCC 26001, ATCC35666, ATCC 29544) and the mixed bacterial solution of the above seven bacteria were incubated at 37°C for 15 min, and the capture efficiency of Phage-MNPs on the above bacterial solutions was determined, and LB solid plates were used for counting.

[0038] The results are as follows Figure 2 As shown in the figure, the capture efficiency of Phage-MNPs for host bacteria ATCC 13076 reached 92.99%, and the capture efficiency for six interfering bacteria: Escherichia coli (ATCC 25922, ATCC 36150), Staphylococcus aureus (ATCC 29213, CMCC26001), Streptococcus dysgalactiae (ATCC 35666) and Cronobacter (ATCC 29544) was less than 15%. In addition, in the seven mixed bacterial solutions of host bacteria and interfering bacteria, the capture efficiency of host bacteria ATCC 13076 can also reach 90.76%, indicating that Phage-MNPs can selectively enrich host bacteria in the presence of multiple interfering bacteria, proving that Phage-MNPs has good specificity.

[0039] Example 3 Phage-MNPs enrichment of host bacteria at different concentrations

[0040] 60 μL Phage-MNPs solution was mixed with different concentrations of Salmonella host bacteria (10 2 -10 6 CFU / mL) (37°C, incubation for 15 min), and the enrichment efficiency of Phage-MNPs at various concentrations was determined. The test results are shown in Figure 3 As shown, Figure 3 In the figure, A and B are the enrichment results in different concentration ranges. It can be seen that this method is effective in the whole 10 2 -10 6 The wide linear range of CFU / mL showed good linear relationship. 2 =0.993. The limit of detection (LOD) is 48 CFU / mL, and the signal-to-noise ratio is 3. The regression equation is y=0.00992x+52.32, where y and x are RLU (fluorescence intensity) and bacterial concentration, respectively. In subsequent practical applications, the bacterial concentration of Salmonella in the sample can be obtained by simply substituting the detected RLU into the above regression equation.

[0041] Example 4 Phage-MNPs combined with ATP bioluminescence to detect Salmonella in skim milk

[0042] Preparation of skim milk samples: A typical single clone of Salmonella ATCC 13076 was selected and inoculated into LB liquid culture medium, cultured overnight at 37°C (180 rpm / min), then 1 mL of the bacterial solution was centrifuged at 5000 rpm for 3 min, washed three times with sterile water, centrifuged again at 5000 rpm for 3 min, and resuspended again with 1 mL of commercially purchased skim milk to obtain a milk sample.

[0043] After adding 60 μL of Phage-MNPs prepared in Example 1 to 1 ml of sample to capture target bacteria (37 ° C, incubation for 15 min), the supernatant was discarded, and 20 μL of 0.05% (mass concentration, the same below) CTAB solution was added and incubated for 3 minutes to quickly lyse the bacteria and release ATP in the bacteria; finally, the enhanced ATP detection kit (purchased from Biyuntian Biotechnology) was used to quantitatively analyze ATP, and finally the linear regression equation y=0.00992x+52.32 was used to calculate the number of bacteria in the solution. At the same time, the traditional dilution coating plate method was used to detect the bacterial content in 1 ml of sample as a control (GB 4789.2-2022 "National Food Safety Standard Food Microbiology Test Colony Count Determination"). The results are shown in Table 1.

[0044] Table 1 Detection results of Salmonella in skim milk

[0045]

[0046] As shown in Table 1, in skim milk, the recovery rate is between 85.43% and 112.58%, and all relative standard deviations (RSD) are lower than 6.61% (the relative standard deviation (RSD) of the ATP detection kit should be less than 10%). This indicates that this method can be used for the detection of Salmonella in skim milk. Figure 4 Schematic diagram of the process of the above detection method.

[0047] Example 5 Phage-MNPs combined with ATP bioluminescence to detect Salmonella in chicken

[0048] Chicken sample preparation: Weigh 2.0g of fresh chicken, irradiate the front and back sides of the chicken with ultraviolet light for 30 minutes in a clean bench for sterilization, and then crush and homogenize until the particle size is less than 5 mm. Add Salmonella ATCC 13076 to the sterile chicken and culture it at 37℃ overnight; the next day, mix the cultured chicken with 18.0mL of sterile water to obtain a chicken sample.

[0049] Then, 1.0 ml of the chicken sample was taken to detect the number of Salmonella using the plate count method and the Phage-MNPs combined with ATP bioluminescence method. The traditional dilution plate method was used as a control (GB 4789.2-2022 "National Food Safety Standard Food Microbiology Test Total Colony Count Determination").

[0050] The above-mentioned Phage-MNPs and ATP bioluminescence coupling method steps are as follows: 60 μL of Phage-MNPs prepared in Example 1 were incubated with chicken samples for 15 min (37 ° C) to capture target bacteria; then the supernatant was discarded, and 20 μL of 0.05% CTAB solution was added and incubated for 3 minutes to quickly lyse the bacteria and release ATP in the bacteria; ATP was quantitatively analyzed using an enhanced ATP detection kit (purchased from Biyuntian Biotechnology), and finally the number of bacteria in the solution was calculated using the linear regression equation y=0.00992x+52.32. The traditional dilution plate method was used as a control (GB 4789.2-2022 "National Food Safety Standard Food Microbiology Test Colony Count Determination"). The results are shown in Table 2.

[0051] Table 2 Detection results of Salmonella in chicken

[0052]

[0053]

[0054] In chicken, the recoveries ranged from 81.50% to 114.97%, and all relative standard deviations (RSDs) were lower than 6.77%, confirming that the assay can be used for the detection of Salmonella in food chicken.

[0055] Example 6 Detection of Listeria monocytogenes in salted duck by combining Phage-MNPs with ATP bioluminescence

[0056] Preparation of salted duck samples: Weigh 2.0 g of salted duck leg meat, irradiate the front and back sides of the duck leg with ultraviolet light for 30 min in a clean bench for sterilization, and then crush and homogenize until the particles are less than 5 mm. Add Listeria monocytogenes (Lm007) to the duck meat and culture it at 37°C overnight, then mix it with 18.0 mL of sterile water to obtain a duck meat sample.

[0057] Functionalized magnetic nanoparticles were prepared using core-shell magnetic nanoparticles and Listeria monocytogenes bacteriophage LipG2-5 (the preparation method was the same as in Example 1, but the bacterial content of the added bacteriophage LipG2-5 was 10 8PFU / mL). 100 μL of functionalized magnetic nanoparticles prepared in this example were incubated with 1.0 ml duck meat sample at 37°C for 15 min to capture the target bacteria; then the supernatant was discarded, and 20 μL of 0.05% CTAB solution was added and incubated for 3 minutes to quickly lyse the bacteria and release ATP in the bacteria; finally, the enhanced ATP detection kit (purchased from Biyuntian Biotechnology) was used to quantitatively analyze ATP, and finally the linear regression equation y=0.0093x+59.78 (the regression equation calculated by the same method as Example 3, y and x are RLU and bacterial concentration, respectively) was used to calculate the number of bacteria in the solution. At the same time, the traditional dilution coating plate method was used to detect the bacterial content of 1.0 ml sample as a control (GB 4789.2-2022 "National Food Safety Standard Food Microbiology Test Colony Count Determination"). The results are shown in Table 3.

[0058] Table 3 Detection results of Listeria monocytogenes in salted duck

[0059]

[0060] As shown in Table 3, the recovery rate of the method of the present application is between 85.10% and 102.80%, and all relative standard deviations (RSD) are lower than 4.17%, indicating that the method can be used for the detection of Listeria monocytogenes in salted duck samples.

[0061] Example 7 Phage-MNPs combined with ATP bioluminescence to detect Staphylococcus aureus in wastewater from dairy farms

[0062] The sewage from the dairy farm was collected and filtered through a 0.22 μm filter membrane. 2 mL of the sample was mixed with 18.0 mL of sterile water to obtain the sewage for later use. A typical monoclonal Staphylococcus aureus CMCC 26001 was selected and inoculated into LB liquid culture medium and cultured overnight at 37°C (180 rpm / min). Then 1 mL of the bacterial solution was centrifuged at 5000 rpm for 3 min, and the mixture was washed three times with sterile water. After centrifugation, 18.0 mL of the sewage obtained in the above step was added to obtain the sewage sample from the farm.

[0063] Functionalized magnetic nanoparticles were prepared using core-shell magnetic nanoparticles and Staphylococcus aureus phage JS25 (the preparation method was the same as in Example 1, except that the Salmonella phage was replaced by the Staphylococcus aureus phage). 80 μL of the functionalized magnetic nanoparticles prepared in this example were incubated with 1 ml of the above-mentioned farm sewage sample for 15 min (37° C.) to capture the target bacteria; then the supernatant was discarded, and 20 μL of 0.05% (mass concentration) CTAB solution was added and incubated for 3 minutes to quickly lyse the bacteria and release ATP in the bacteria; finally, an enhanced ATP detection kit (purchased from Biyuntian Biotechnology) was used to quantitatively analyze ATP, and finally the number of bacteria in the solution was calculated using the linear regression equation y=0.0105x+48.69 (the regression equation calculated using the same method as in Example 3, y and x are RLU and bacterial concentration, respectively).

[0064] At the same time, the traditional dilution plate method was used to detect the bacterial count of 1 ml sample as a control (GB 4789.2-2022 "National Food Safety Standard Food Microbiology Test Total Colony Count Determination"). The results are shown in Table 4.

[0065] Table 4 Detection results of Staphylococcus aureus in wastewater from dairy farms

[0066]

[0067] As shown in Table 4, the recovery rate of the detection method of the present application is between 89.56% and 105.03%, and all relative standard deviations (RSD) are lower than 3.16%, indicating that the method can be used for the detection of Staphylococcus aureus in environmental samples.

[0068] Example 8 Phage-MNPs combined with ATP bioluminescence to detect Staphylococcus aureus in bovine serum

[0069] Preparation of serum samples: The bovine serum sample was first filtered through a 0.22 μm filter membrane, and then 2 mL of the sample was mixed with 18.0 mL of sterile water as a serum-containing matrix sample for later use; a typical monoclonal Staphylococcus aureus CMCC 26001 was selected and inoculated into LB liquid culture medium, cultured overnight at 37°C (180 rpm), and then 1 mL of the bacterial solution was centrifuged at 5000 rpm for 3 min, washed three times with sterile water, and centrifuged again. 18.0 mL of the serum-containing matrix sample prepared by the above method was added and resuspended to obtain a serum sample.

[0070] 80 μL of the functionalized magnetic nanoparticles prepared in Example 7 were incubated with 1.0 ml of the serum sample for 15 min (37° C.) to capture the target bacteria; the supernatant was then discarded, and 20 μL of 0.05% (mass concentration) CTAB solution was added and incubated for 3 minutes to quickly lyse the bacteria and release ATP in the bacteria; finally, an enhanced ATP detection kit (purchased from Beyotime Biotechnology) was used to quantitatively analyze ATP, and finally the linear regression equation y=0.0105x+48.69 was used to calculate the number of bacteria in the solution.

[0071] At the same time, the traditional dilution plate method was used to detect the bacterial count of 1.0 ml sample as a control (GB 4789.2-2022 "National Food Safety Standard Food Microbiology Test Total Colony Count Determination"). The results are shown in Table 5.

[0072] Table 5 Detection results of Staphylococcus aureus in bovine serum samples

[0073]

[0074] As shown in Table 5, the recovery rate of the method of the present application is between 88.14% and 106.30%, and all relative standard deviations (RSD) are lower than 3.75%, indicating that the method can be used for the detection of Staphylococcus aureus in serum samples contaminated by Staphylococcus aureus in clinical samples.

Claims

1. A rapid and accurate detection method for pathogenic bacteria, characterized in that: The specific steps are as follows: 1) Take 5 mg of core-shell magnetic nanoparticles and place them in a centrifuge tube. Incubate them with alcohol and MES in sequence. Then use a magnet to adsorb the magnetic nanoparticles to the tube wall and remove the supernatant. Then, 1 mL of the modifier was added to the centrifuge tube, and the tube was ultrasonically treated for 30 min and shaken at 600 rpm for 1 h. The magnetic nanoparticles were adsorbed on the tube wall using a magnet again, and the supernatant was removed. Add an equal volume of PBS to the centrifuge tube for washing, and then add 1 mL of 10 8 ~10 12 PFU / mL phage, 180rpm, incubate at 37°C for 12 h; the phage is the phage corresponding to the target bacteria; The centrifuge tube was washed again with PBS, 500 μL of 5.0% BSA solution was added, and the mixture was reacted at 180 rpm for 30 min. The magnetic nanoparticles were adsorbed on the tube wall using a magnet, and the BSA solution was discarded. The centrifuge tube was then washed three times with 1 mL of PBS, and finally resuspended with 1 mL of PBS to obtain functionalized magnetic nanoparticles. The preparation method of the modifier is as follows: add EDC with a final concentration of 200 mg / mL and NHS with a final concentration of 100 mg / mL to a 25 mM MES solution, and then filter with a filter membrane with a pore size of 0.22 µm to obtain the modifier; 2) Take 1 ml of the sample to be tested and incubate it with 60-100 μL of the functionalized magnetic nanoparticles obtained in step 1) for 15 min; 3) Discard the supernatant in the solution, add 20 µL of 0.05% CTAB solution and incubate for 3 minutes; finally, use the ATP detection kit to detect the fluorescence intensity of ATP in the solution, and substitute it into the regression equation corresponding to the target bacteria to obtain the viable bacterial content of the target bacteria in the sample.

2. The rapid and accurate detection method for pathogenic bacteria according to claim 1, characterized in that: The target bacteria is one of Salmonella, Listeria, and Staphylococcus aureus.

3. The rapid and accurate detection method for pathogenic bacteria according to claim 1, characterized in that: The sample is liquid or solid; if the sample is solid, 2 g of the sample is crushed to a particle size of less than 5 mm, and then 18 ml of sterile water is added to prepare a mixed solution, which is the sample to be tested.

4. The rapid and accurate detection method for pathogenic bacteria according to claim 2, characterized in that: When the target bacteria is Salmonella, the regression equation is y=0.00992x+52.32; when the target bacteria is Listeria, the regression equation is y=0.0093x+59.78; when the target bacteria is Staphylococcus aureus, the regression equation is y=0.0105x+48.69; the above y and x are fluorescence intensity and bacterial concentration respectively.

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