CN103217530B - NMR food-borne pathogen rapid detection method based on paramagnetic nano-Fe-Co alloy probe indirect enrichment - Google Patents

Abstract

The invention discloses an NMR rapid detection method for food-borne pathogenic bacteria based on indirect enrichment of paramagnetic nanometer Fe-Co alloy probes, belonging to the technical field of rapid detection of food-safe pathogenic bacteria. The present invention relies on the established nuclear magnetic resonance detection method that can be used for pathogenic bacteria in food liquid samples, uses the first antibody to capture the target bacteria, and utilizes the antibody of the first antibody, that is, the paramagnetic nano-Fe-Co alloy coated with the second antibody The probe enriches and separates the target bacteria, and uses the influence of the paramagnetic properties of the nano-Fe-Co alloy on the relaxation time of the nuclear magnetic resonance attenuation signal to detect whether the sample contains the target pathogenic bacteria. The different specific corresponding relationships are: the paramagnetic nano-Fe-Co alloy probe shows a linear relationship under certain conditions, that is, the larger the content of the nano-Fe-Co alloy, the smaller the T2/T1 value of the sample, and it can quantify within a certain range Detect target bacteria. The method can be used for rapid detection of harmful pathogenic bacteria in food samples, and thus can be used as a rapid screening of a large number of samples to be tested.

Description

A NMR rapid detection method for foodborne pathogens based on indirect enrichment of paramagnetic nano-Fe-Co alloy probes

technical field

The invention relates to a rapid detection method for pathogenic bacteria, in particular to an NMR rapid detection method for food-borne pathogenic bacteria based on the indirect enrichment of paramagnetic nanometer Fe-Co alloy probes.

Background technique

Basic principle: Monoclonal antibodies or antigen molecules are combined with enzyme molecules through covalent bonds. This combination will not change the immunological characteristics and biological activities of monoclonal antibodies, antigens and enzymes. Specific monoclonal antibodies will only interact with specificity. antigen binding. Fe-Co alloy materials are ferromagnetic, and paramagnetic properties will appear when the particle diameter is small enough to a certain extent. That is, there is no magnetism when there is no external magnetic field, but it shows certain magnetism when there is an external magnetic field, which can be used for magnetic separation. At the same time, the influence of paramagnetic substances on NMR signals is very significant, and a small amount of paramagnetic substances will cause changes in NMR signals. Therefore, a paramagnetic specific Fe-Co alloy nanoprobe biosensor can be constructed for detection from the perspective of magnetic resonance.

The main principle steps: 1. Add the first antibody to detect the target bacteria in the sample. If the target bacteria exists in the sample, the first antibody complex will be formed through the combination of antibody and antigen. At this time, the first antibody will amplify the signal. 2. Purchase paramagnetic nano-Fe-Co alloy materials from the market, or prepare nano-scale Fe-Co alloys by other methods. Use silane coupling agent, its general formula is: Y(CH ₂ )nSiX ₃ . Here, n is 0-3; X is a hydrolyzable group; Y is an organic functional group. X is usually chlorine group, methoxy group, ethoxy group, acetoxy group, etc. When these groups are hydrolyzed, they will generate silanol (Si(OH) ₃ ), and combine with inorganic substances to form siloxane. Y is a vinyl group, an amino group, an epoxy group, a methacryloxy group, or a mercapto group. These reactive groups can react with organic substances to combine. Therefore, by using silane coupling agent, a "molecular bridge" can be built between the interface of inorganic substances and organic substances, and the two materials with different properties can be connected together to improve the performance of composite materials and increase the bonding strength. Surface functionalization can be achieved by modifying antibodies to form specific immune probes and then block redundant active sites. Due to the paramagnetic properties of the nano-Fe-Co alloy probe, the unbound antibody can be separated by an external magnetic field. The nano-Fe-Co alloy material is modified with the antibody of the first antibody, that is, the second antibody. 3. Use a certain method to immobilize the target bacteria-specific first antibody on the surface of the solid phase carrier, and seal the redundant active sites for future use. 4. Add the paramagnetic nano-Fe-Co alloy probe prepared in the second step to the sample treated in the first step, fully mix and oscillate for a period of time, and apply an external magnetic field after grabbing the target bacteria. Since the nano-Fe-Co alloy With paramagnetic properties, the Fe-Co alloy probes will gather to the side of the magnetic field, and the probes can be separated by sucking away the supernatant. If there are target bacteria in the sample to be tested, it will first form a 1-antibody complex with the 1-antibody in the first step, and the 1-antibody complex will then complex with the 2-antibody on the surface of the probe, and be enriched and separated by an external magnetic field . After washing and magnetic separation, a small amount of sterile deionized water is added to form a paramagnetic nano-Fe-Co alloy probe suspension of the target bacteria. At this time, the excess probes that caught the target bacteria and those that did not catch the target bacteria were still mixed together. 5. Add the above-mentioned mixed probes to the surface of the solid-phase carrier in step 3, and the probes that have captured the target bacteria will specifically bind to the monoclonal antibody on the surface of the solid-phase carrier to form a double-antibody sandwich. Rinse with sterile deionized water to elute unbound probes. 6. Then use the eluent to wash off the bound specific nano-immunoprobes on the immobilized carrier, and use the magnetic separation method to wash away the ions and solvents. If this part of the probe exists, it is the probe that has captured the target bacteria. Due to the paramagnetic properties of Fe-Co alloys, they are very sensitive to resonance instruments. Compared with other molecules, a small amount of Fe-Co alloys can greatly reduce the spin-lattice relaxation parameters T1 and Spin-spin relaxation time T2, and sterile deionized water under a certain uniform field strength, T1/T2 is fixed. The eluate was placed in a nuclear magnetic resonance apparatus, and compared with a sterile deionized water control group. A significant decrease in T1/T2 value indicates the existence of the probe, which indirectly indicates the detection of pathogenic bacteria in the food sample. Probe content was proportional to the decrease in T1/T2 value. By adding standard, the target bacteria can be quantitatively detected. In this method, the Fe-Co alloy probe is not only a means for separation and enrichment, but also the Fe-Co alloy has paramagnetic properties and can be used as a probe for quantitative detection. The main advantages of this method are rapidity and high sensitivity. Compared with the microbial culture of pathogenic bacteria, it takes 2-3 days or even several days. This method mainly depends on the pretreatment time of the sample, and NMR detection takes only a few minutes. All food standards, pathogenic bacteria must not be detected. Therefore, this method can be used for positive screening of large-scale samples to be tested, and quantitative detection can be performed to a certain extent. At present, there is no literature reporting this method at home and abroad.

Contents of the invention

A rapid NMR method for the detection of foodborne pathogens based on the indirect enrichment of paramagnetic nano-Fe-Co alloy probes for the evaluation of various food samples. This method is an objective and effective method for detecting harmful pathogenic bacteria in food, thereby greatly reducing the screening time of harmful pathogenic bacteria in food samples to some extent.

A rapid NMR method for the detection of food-borne pathogens based on the indirect enrichment of paramagnetic nano-Fe-Co alloy probes. Using the sensitivity of the NMR instrument to paramagnetic substances, the relationship between the change of the NMR relaxation parameters and the Fe-Co alloy was proposed. Correlation indicators for the content of paramagnetic immunoprobes in Co alloy nanoparticles. Different pathogens have different detection limits.

This method relies on the enrichment and separation of specific paramagnetic nano-Fe-Co alloy probes that can be used for harmful pathogenic bacteria in food samples, and detects harmful pathogenic bacteria in samples from the perspective of changes in NMR relaxation signal parameters. bacteria. The specific pathogenic bacteria in the sample can be enriched by using the paramagnetic nanometer Fe-Co alloy probe coupled with the specific monoclonal antibody. Since the spin-lattice relaxation efficiency and spin-spin relaxation efficiency of the NMR instrument are very sensitive to Fe-Co alloy nanoparticles, that is, in deionized water, there are traces of paramagnetic Fe-Co alloy nanoparticles, then The spin-lattice relaxation time (T1) and/or spin-spin relaxation (T2) of water will decrease significantly. Under certain conditions, the paramagnetic properties of the paramagnetic immunoprobes lead to a linear decrease in the relaxation decay signal T2/T1 of the NMR. By quantitatively detecting the content of the immune probe in the sample, the content of harmful pathogenic bacteria in the food sample can be indirectly quantified. The detected pathogenic bacteria content was linearly correlated with the probe content, and the fitting degree was good. Finally, the number of pathogenic bacteria colonies in the food samples was determined based on the corresponding relationship between the paramagnetic immunoprobe and the pathogenic bacteria. And all food standards, pathogenic bacteria must not be detected. Therefore, this method can be used for positive screening of large-scale samples to be tested, and quantitative detection to a certain extent.

The present invention is achieved like this, and the steps are as follows:

1) Add the first antibody to detect the target bacteria in the sample. If the target bacteria exists in the sample, the first antibody complex will be formed through the combination of antibody antigen;

2) Preparation of the antibody for detecting the first antibody of the target bacteria, that is, the second antibody-coated paramagnetic nano-Fe-Co alloy probe;

3) Immobilize the target bacteria-specific first antibody on the solid phase carrier for later use;

4) Enrich and isolate the target strain: add the paramagnetic nano-Fe-Co alloy probe prepared in the second step to the sample treated in the first step, mix thoroughly and oscillate for a period of time, grab the target bacteria and apply additional Magnetic field, due to the paramagnetic properties of the nano-Fe-Co alloy, the Fe-Co alloy probes will be gathered to the side of the magnetic field, and the probes can be separated by sucking the supernatant. If there are target bacteria in the sample to be tested, it will first form a 1-antibody complex with the 1-antibody in the first step, and the 1-antibody complex will then complex with the 2-antibody on the surface of the probe, and be enriched and separated by an external magnetic field . After washing and magnetic separation, a small amount of sterile deionized water is added to form a paramagnetic nano-Fe-Co alloy probe suspension of the target bacteria. At this time, the excess probes that caught the target bacteria and those that did not catch the target bacteria were still mixed together.

5) Add the enriched probe suspension to the solid-phase carrier immobilized with the monoclonal antibody prepared in step 3. If there are target bacteria, a double-antibody sandwich will be formed; wash with sterile deionized water, there will be no The probes that capture the target bacteria are washed off, and if there are no target bacteria, all probes are washed off;

6) After that, wash off the double-antibody sandwich probe on the fixed plate with an eluent, and use an external magnetic field to separate the probe and wash off ions and solvents with sterile deionized water. If the probe still exists, it is caught probes to target bacteria. For this part of the probe, add sterile deionized water to form a suspension of the probe, and measure the relaxation time of the nuclear magnetic resonance. Using sterile deionized water as a blank, the measured relaxation time T1 of the suspension is /T2 is significantly lower than that of sterile deionized water, indicating that the probe is contained, thus indirectly proving that there are target pathogenic bacteria in the sample. To a certain extent, the amount of target bacteria can be quantified indirectly.

The NMR probe is a nanoscale Fe-Co alloy material with paramagnetic properties, and the nanometer particle diameter is less than 1000 nanometers.

The final detection and evaluation method of the target bacteria is based on the change of the relaxation time characteristic parameter of the nuclear magnetic resonance technique.

The relaxation time characteristics refer to spin-lattice relaxation time (T1) and spin-spin relaxation time (T2).

For the relaxation time characteristics, T1 is measured by the 180˚-τ-90˚ pulse method, and T2 is measured by the CPMG sequence method.

The primary antibody is a specific antibody for detecting target bacteria, preferably a monoclonal antibody. Antibody 2 is the antibody of the first antibody.

Beneficial effect of the present invention: the present invention provides a kind of method that the harmful pathogenic bacteria in food is detected rapidly objectively, it is characterized in that relying on the NMR that can be used to detect the indirect enrichment of paramagnetic nanometer Fe-Co alloy probe Resonance detection method. This method can objectively and effectively detect harmful pathogenic bacteria in food. Compared with the biological culture confirmation of pathogenic bacteria, this method has the advantage of rapid detection and can be used for rapid screening of large-scale samples.

Detailed ways

Example 1

Test food samples for the presence of the harmful pathogen Listeria monocytogenes.

1. Nano-Fe-Co alloy immunoprobe preparation: the first antibody is rabbit anti-IgG monoclonal antibody of Listeria monocytogenes, and the second antibody is goat anti-rabbit IgG of Listeria monocytogenes. Fe-Co alloy nanoparticles can be purchased from the market. For example, Beijing Deke Daojin Technology Co., Ltd. or Shanghai Chaowei Nanomaterials Co., Ltd., 20nm, ratio 1:1, each with a purity of 99.5%.

Silica-coated nano-Fe-Co alloy: take 47.5g of sodium silicate, dissolve it in a beaker with deionized water, and adjust the pH value to 12-13 with hydrochloric acid. Take 5.0g of nano-Fe-Co alloy and add it into the beaker, and stir mechanically (with a glass rod) for 5min. The mixture was sonicated for 30 min and stirred in due course. Raise the temperature to 85˚C, add hydrochloric acid dropwise to adjust the pH value to 6-7, and form a precipitate. While magnetically separating, wash the precipitate with deionized water for 3-4 times. Then, the precipitate was dispersed in 250 mL of methanol. The above process is repeated three times to ensure that the silicon is attached to the Fe-Co alloy.

Amine-based silylated Fe-Co alloy nanomaterials: The prepared silica-coated nano-Fe-Co alloy was added to 25 mL of methanol, and diluted to 150 mL with 1 mL of H ₂ O and methanol. Then 150 mL of glycerin was added and mixed. Sonicate for 30min, and transfer to a 500mL three-necked flask with a stirring device. Add 10 mL of aminosilane coupling agent (AEAPS), and after rapid stirring at 80-90 ˚C for 3 h, the product is transferred. The product was washed 3 times with deionized water and 2 times with methanol (suction filtration with a Buchner funnel). Vacuum dry. It should be noted that the suction filtration is relatively slow due to the presence of glycerin. After a period of suction, the upper liquid can be sucked off with a straw in due course. The suction filtration process takes about 6-8 hours. Finally, the obtained aminosilylated Fe—Co alloy nanomaterials were dried in vacuum for 12 hours.

Antibody modification: Take a certain amount of aminated Fe-Co alloy nanoparticles, add excess goat anti-rabbit IgG antibody against Listeria monocytogenes, incubate at 26 °C, wash, add excess 1% bovine serum albumin (BSA), 22 ˚C, seal surface active vacancies, wash, resuspend. Due to the paramagnetic properties of nano-Fe-Co alloys, when an external magnetic field is applied to separate them, nano-Fe-Co alloys will gather to the side of the magnetic field, absorb the supernatant, and wash away excess antibodies and BSA. The prepared paramagnetic nano-immunoprobes were stored at 4 °C until use.

2. Monoclonal antibody immobilization: You can use the conventional enzyme plate immobilization method, or you can use the following methods. Use a clean coverslip 5×5mm ^square , spray a layer of Cr (2–4 nm) with a coater to help fix the gold. Then spray a layer of nano-gold on the surface by sputtering, and then use 200 microliters of 2 mmol dithio-succinimide-propionate (DSP) to modify the nano-gold (DMSO, dimethyl sulfoxide diluted DSP ). Add the primary antibody of Listeria monocytogenes and rabbit anti-IgG monoclonal antibody, that is, 100 µL of 100 µg/mL monoclonal antibody is fixed on the glass plate by covalent coupling method and incubated at 37 °C for 45 min. Add 1% bovine serum albumin (BSA), 22˚C, 1 hour, the remaining active sites on the plate are blocked and dried.

3. Pre-treat the food samples, and if necessary, use the FDA enrichment method to filter, enrich and activate the samples to obtain the samples to be tested. The first antibody, rabbit anti-IgG against Listeria monocytogenes, was added to the sample to be tested. If Listeria monocytogenes exists in the sample to be tested, it will form a 1-antibody complex with 1 antibody. After adding the goat anti-rabbit IgG probe of the second antibody Listeria monocytogenes prepared in step 1, shake fully. Separate the probe on a magnetic stand, and add a small amount of sterile deionized water to obtain a probe suspension. At this time, if the target Listeria monocytogenes exists in the sample to be tested, the complex is captured through the interaction between the second antibody and the first antibody, and the purpose of enriching the target bacteria is achieved. At this time, both the 1-antibody complex bound to the target bacterium and the excess 1 antibody not bound to the target bacterium will bind to the 2-antibody on the surface of the probe, or be mixed together. Add this enriched probe suspension to the 1-antibody immobilization plate prepared in step 2, then the probes combined with Listeria monocytogenes in the probe suspension will further combine with the monoclonal Antibodies are specifically combined to form a double-antibody sandwich structure. At this time, wash with sterile deionized water to wash away the probes that are not bound to Listeria monocytogenes. All that remains on the fixed plate is the L. monocytogenes-bound probe.

4. Use an eluent (methanol, etc.) to elute the probe bound to Listeria monocytogenes on the fixed plate. Put on the magnetic stand, separate the probe and wash it 1-2 times to wash away the ions. The obtained solution was used to measure T1 and T2 of the solution with a nuclear magnetic resonance instrument (NMR20, Numei Company). Using sterile deionized water as a blank, there is a significant difference between the T1/T2 measured in the solution and the blank, indicating that there is a probe in the solution, thereby indicating that there is Listeria monocytogenes in the sample. The amount of probe is proportional to the decrease in T1/T2. The greater the decrease, the more probes there are, which indirectly indicates the more Listeria monocytogenes. The number of target bacteria in the sample can be quantitatively detected by standard addition verification. All food standards must not detect Listeria monocytogenes, and this method can quickly detect whether a sample contains Listeria monocytogenes.

Example 2

Determination of food samples for the presence of harmful pathogenic bacteria Escherichia coli O157:H7.

1. Preparation of nano-Fe-Co alloy immunoprobes: 1st antibody uses O157:H7 rabbit anti-IgG monoclonal antibody, 2nd antibody is O157:H7 goat anti-rabbit IgG, which can be monoclonal antibody or polyclonal antibody. Fe-Co alloy nanoparticles can be purchased from the market. For example, Beijing Deke Daojin Technology Co., Ltd. or Shanghai Chaowei Nanomaterials Co., Ltd., 20nm, ratio 1:1, each with a purity of 99.5%.

Silica-coated nano-Fe-Co alloy: take 47.5g of sodium silicate, dissolve it in a beaker with deionized water, and adjust the pH value to 12-13 with hydrochloric acid. Take 5.0g of nano-Fe-Co alloy and add it into the beaker, and stir mechanically (with a glass rod) for 5min. The mixture was sonicated for 30 min and stirred in due course. Raise the temperature to 85˚C, add hydrochloric acid dropwise to adjust the pH value to 6-7, and form a precipitate. While magnetically separating, wash the precipitate with deionized water for 3-4 times. Then, the precipitate was dispersed in 250 mL of methanol. The above process is repeated three times to ensure that the silicon is attached to the Fe-Co alloy.

Amine-based silylated Fe-Co alloy nanomaterials: The prepared silica-coated nano-Fe-Co alloy was added to 25 mL of methanol, and diluted to 150 mL with 1 mL of H ₂ O and methanol. Then 150 mL of glycerin was added and mixed. Sonicate for 30min, and transfer to a 500mL three-necked flask with a stirring device. Add 10 mL of aminosilane coupling agent (AEAPS), and after rapid stirring at 80-90 ˚C for 3 h, the product is transferred. The product was washed 3 times with deionized water and 2 times with methanol (suction filtration with a Buchner funnel). Vacuum dry. It should be noted that the suction filtration is relatively slow due to the presence of glycerin. After a period of suction, the upper liquid can be sucked off with a straw in due course. The suction filtration process takes about 6-8 hours. Finally, the obtained aminosilylated Fe—Co alloy nanomaterials were dried in vacuum for 12 hours.

Antibody modification: take a certain amount of aminated Fe-Co alloy nanoparticles, add an excess of 2 antibodies, that is, goat anti-rabbit IgG antibody of Escherichia coli O157:H7, incubate at 26 °C, wash, and add an excess of 1% bovine serum albumin ( BSA), 22˚C, seal surfactant vacancies, wash, resuspend. Due to the paramagnetic properties of nano-Fe-Co alloys, when an external magnetic field is applied to separate them, nano-Fe-Co alloys will gather to the side of the magnetic field, absorb the supernatant, and wash away excess antibodies and BSA. The prepared paramagnetic nano-immunoprobes were stored at 4 °C until use.

2. Monoclonal antibody immobilization: You can use the conventional enzyme plate immobilization method, or you can use the following methods. Use a clean coverslip 5×5mm ^square , spray a layer of Cr (2–4 nm) with a coater to help fix the gold. Then spray a layer of nano-gold on the surface by sputtering, and then use 200 microliters of 2 mmol dithio-succinimide-propionate (DSP) to modify the nano-gold (DMSO, dimethyl sulfoxide diluted DSP ). Add the first antibody, that is, rabbit anti-IgG monoclonal antibody, that is, 100 μL of 100 μg/mL monoclonal antibody is fixed on the glass plate by covalent coupling method and incubated at 37 °C for 45 min. The remaining active sites on the plate were blocked by adding bovine serum albumin and dried.

3. Pre-treat the food samples, and if necessary, use the FDA enrichment method to filter, enrich and activate the samples to obtain the samples to be tested. Add the first antibody, O157:H7 rabbit anti-IgG, to the sample to be tested. If O157:H7 exists in the sample to be tested, it will form a 1-antibody complex with 1 antibody. Add the second antibody prepared in the first step, the goat anti-rabbit IgG probe of O157:H7, and shake it sufficiently. Separate the probe on a magnetic stand, and add a small amount of sterile deionized water to obtain a probe suspension. At this time, if the target Listeria monocytogenes exists in the sample to be tested, the complex is captured through the interaction between the second antibody and the first antibody, and the purpose of enriching the target bacteria is achieved. At this time, both the 1-antibody complex bound to the target bacterium and the excess 1 antibody not bound to the target bacterium will bind to the 2-antibody on the surface of the probe, or be mixed together. Add this enriched probe suspension to the 1-antibody immobilization plate prepared in step 2, then the probes combined with O157:H7 in the probe suspension will further react with the monoclonal antibody on the immobilization plate Specific binding to form a double-antibody sandwich structure. At this time, wash with sterile deionized water to wash away the probes that are not bound to O157:H7. All that remains on the fixed plate is the O157:H7-bound probe.

4. Use the eluent (methanol, etc.) to elute the probe bound to E. coli O157:H7 on the fixed plate. Put on the magnetic stand, separate the probe and wash it 1-2 times with sterile deionized water to remove ions and solvents. The solution that obtains, measures the T1 and T2 of solution with nuclear magnetic resonance instrument (NMR20, Newmay company or miFe-Co alloy NMR East China Normal University), takes deionized water as blank, and the T1/T2 that solution records compares with blank, has A significant difference indicates that there is a probe in the solution, thereby indicating that there is Escherichia coli O157:H7 in the sample, and the amount of the probe is directly proportional to the decrease value of T1/T2. The greater the decrease, the more probes, which indirectly indicates the more E. coli O157:H7, and the number of target bacteria in the sample can be quantitatively detected through standard addition verification.

Claims (6)

1. A NMR food-borne pathogen rapid detection method based on paramagnetic nano Fe-Co alloy probe indirect enrichment, its characteristic steps are as follows: 1) Add a specific primary antibody to detect the target bacteria in the sample. If the target bacteria exists in the sample, the primary antibody complex will be formed through the combination of the antibody antigen; 2) The antibody for detecting the primary antibody of the target bacteria, that is, the preparation of the secondary antibody-coated paramagnetic nano-Fe-Co alloy probe; 3) Immobilize the target bacteria-specific primary antibody on the fixed plate for later use; 4) Enrichment and separation of target strains: Add the paramagnetic nano-Fe-Co alloy probe prepared in step 2 to the sample treated in step 1, mix well and oscillate for a period of time, grab the target bacteria and apply With an external magnetic field, due to the paramagnetic properties of nano-Fe-Co alloys, the Fe-Co alloy probes will gather to the side of the magnetic field, and the probes can be separated by absorbing the supernatant; In the first step, a primary antibody complex is formed with the primary antibody, and the primary antibody complex will then complex with the secondary antibody on the surface of the probe, and be enriched and separated by applying an external magnetic field; after washing and magnetic separation, add a small amount of sterile deionized The ionized water forms the paramagnetic nano-Fe-Co alloy probe suspension of the target bacteria; at this time, the Fe-Co alloy probes that capture and do not capture the primary antibody complex are still mixed together; 5) Add the enriched Fe-Co alloy probe suspension to the fixed plate on which the monoclonal antibody was immobilized in step 3. If there are target bacteria, a double-antibody sandwich will be formed, and washed with sterile deionized water, then The probes that do not capture the target bacteria are washed away, and if there are no target bacteria, all probes are washed away; 6) After that, wash off the double-antibody sandwich probe on the fixed plate with an eluent, and use an external magnetic field to separate the probe and wash off ions and solvents with sterile deionized water. If the probe still exists, it is caught Probes to target bacteria; 7) For this part of the probe, add sterile deionized water to form a probe suspension, and perform NMR relaxation time measurement; Under a fixed field strength, the T1 or T2 of sterile deionized water is a constant value. Taking sterile deionized water as a blank, the measured relaxation time T1 or T2 of the suspension is compared with that of sterile deionized water. A significant decrease in ionized water indicates that it contains probes, which indirectly proves that there are target pathogenic bacteria in the sample. The amount of probes is directly proportional to the decrease of T1 or T2, and the amount of target bacteria can be indirectly quantified by adding standard quantitative probes . 2. the NMR foodborne pathogen rapid detection method based on the indirect enrichment of paramagnetic nanometer Fe-Co alloy probe according to claim 1, is characterized in that described probe is the nanometer Fe-Co alloy with paramagnetic property Co alloy material. 3. the NMR food-borne pathogen rapid detection method based on paramagnetic nano Fe-Co alloy probe indirect enrichment according to claim 1, it is characterized in that the final detection evaluation method of described target bacteria is based on NMR Variation of the relaxation time parameter of the resonance technique. 4. the NMR food-borne pathogen rapid detection method based on paramagnetic nano Fe-Co alloy probe indirect enrichment according to claim 1 is characterized in that described relaxation time refers to spin-lattice Relaxation time (T1) and spin-spin relaxation time (T2). 5. The NMR food-borne pathogen rapid detection method based on paramagnetic nano-Fe-Co alloy probe indirect enrichment according to claim 1, is characterized in that, T1 adopts 180˚-τ-90˚ pulse method to measure , T2 was determined by CPMG sequence method. 6. the NMR food-borne pathogen rapid detection method based on the indirect enrichment of paramagnetic nanometer Fe-Co alloy probe according to claim 1, is characterized in that described primary antibody is the specific monoclonal of detection target bacterium Cloned antibody, the secondary antibody is the antibody of the primary antibody.