CN103226116B - A kind of NMR food-borne pathogen rapid detection based on indirect enrichment of paramagnetic nano-Co probe - Google Patents

Abstract

The invention discloses an NMR rapid detection method for food-borne pathogenic bacteria based on the indirect enrichment of paramagnetic nano-Co 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, utilizes 1 antibody to capture target bacteria, and utilizes the antibody of 1 antibody, that is, the paramagnetic nano-Co probe prepared by 2 anti-coating to target bacteria Enrichment and separation are carried out, and the effect of the paramagnetic properties of nano-Co on the relaxation time of the nuclear magnetic resonance attenuation signal is used to detect whether the sample contains target bacteria. The different specific correspondences are: paramagnetic nano-Co probes show a linear relationship under certain conditions, that is, the content of nano-Co is large, and the spin-lattice relaxation time and spin-spin relaxation time of the sample1 The smaller the value, the quantitative detection of target bacteria can be carried out within a certain range. 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 rapid NMR method for the detection of foodborne pathogens based on indirect enrichment of paramagnetic nano-Co 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 nano-Co 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. Co material is 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 Co nanoprobe biosensor can be constructed for detection from the perspective of magnetic resonance.

The main principle steps: 1. Add the 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 antibody and antigen, and the primary antibody will amplify the signal at this time. 2. Purchase paramagnetic nano-Co materials from the market, or prepare nano-scale Co 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-Co probes, unbound antibodies can be separated by an external magnetic field. The nano-Co material is modified with the antibody of the 1st antibody, that is, the 2nd antibody. 3. Use a certain method to immobilize the target bacterium-specific 1 antibody on the surface of the microtiter plate, and seal the redundant active sites for future use. 4. Add the paramagnetic nano-Co 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. Due to the paramagnetic properties of nano-Co, Co The probes will gather to one side of the magnetic field, and the probes can be separated by aspirating 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-Co 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 microtiter plate in step 3, and the probe that has captured the target bacteria will specifically bind to the monoclonal antibody on the surface of the microtiter plate 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 Co, it is very sensitive to resonance instruments. Compared with other molecules, a small amount of Co can greatly reduce the spin-lattice relaxation parameter T1 and spin-spin relaxation of sterile deionized water. Time T2, while the sterile deionized water is under a certain uniform field strength, the spin-lattice relaxation time and the spin-spin relaxation time are fixed. The eluate was placed in a nuclear magnetic resonance apparatus, and compared with a sterile deionized water control group. Significant decrease in spin-lattice relaxation time and spin-spin relaxation time indicates the presence of probes, which indirectly indicates that pathogenic bacteria have been detected in food samples. Probe content is proportional to spin-lattice relaxation time and spin-spin relaxation time value decrease. By adding standard, the target bacteria can be quantitatively detected. In this method, the Co probe is not only a means of separation and enrichment, but also Co 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 indirect enrichment of paramagnetic nano-Co 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-Co probes. Using the sensitivity of NMR to paramagnetic substances, the relationship between the change of NMR relaxation parameters and the paramagnetic properties of Co nanoparticles is proposed. Correlation index for immune probe content. Different pathogens have different detection limits.

The method relies on the enrichment and separation of specific paramagnetic nano-Co probes that can be used for harmful pathogenic bacteria in food samples, and detects harmful pathogenic bacteria in samples from the perspective of NMR relaxation signal parameter changes. The specific pathogenic bacteria in the sample can be enriched by using the paramagnetic nano-Co probe coupled with the specific monoclonal antibody. Because the spin-lattice relaxation efficiency and spin-spin relaxation efficiency of NMR are very sensitive to Co nanoparticles, that is, in deionized water, there are traces of paramagnetic Co nanoparticles, and the spin-lattice The lattice relaxation time (T1) and/or spin-spin relaxation (T2) will decrease significantly. Under certain conditions, the paramagnetic properties of paramagnetic immunoprobes lead to a linear decrease in the spin-lattice relaxation time and spin-spin relaxation time of the NMR relaxation decay signal. 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 food samples was determined based on the corresponding relationship between paramagnetic immunoprobes and 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 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 antibody and antigen.

2) The preparation of the antibody for detecting the 1-antibody of the target bacteria, that is, the 2-antibody-coated paramagnetic nano-Co probe;

3) Immobilize the target bacteria-specific 1 antibody on the microtiter plate for later use.

4) Enrich the target bacteria and separate them: Add the paramagnetic nano-Co probe prepared in the second step to the sample treated in the first step, mix and shake for a period of time, and apply an external magnetic field after grabbing the target bacteria. Due to the paramagnetic properties of nano-Co, the Co probes will converge 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-Co 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 microtiter plate immobilized with 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 away, and if there are no target bacteria, all probes are washed away.

6) Afterwards, wash off the double-antibody sandwich probes on the ELISA plate with an eluent, and use an external magnetic field to separate the probes and wash off ions and solvents with sterile deionized water. If there are still probes Probes that capture target bacteria. For this part of the probe, add sterile deionized water to form the suspension of the probe, and carry out the measurement of the relaxation time of nuclear magnetic resonance. With sterile deionized water as a blank, the relaxation time of the measured suspension is automatically If the spin-lattice relaxation time and spin-spin relaxation time are significantly lower than those in sterile deionized water, it indicates that the probe is contained, thus indirectly proving that there are target bacteria in the sample. The amount of the probe is related to the spin -The decrease of the lattice relaxation time and the spin-spin relaxation time is directly proportional, and the amount of the target bacteria can be indirectly quantified to a certain extent through the quantitative probe.

The NMR probe is a nanoscale Co material with paramagnetic properties, and the nanometer particle size 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 and spin-spin relaxation time.

The primary antibody is a specific antibody for detecting target bacteria, preferably a monoclonal antibody. The 2-antibody is the antibody of the 1-antibody.

Beneficial effect of the present invention: the present invention provides a kind of method that objectively detects the harmful pathogenic bacteria in the food quickly, it is characterized in that relying on the nuclear magnetic resonance detection method that can be used to detect the indirect enrichment of paramagnetic nanometer Co probe . 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 description

Example 1

Examine food samples to determine whether they contain the harmful pathogen Listeria monocytogenes.

1. Preparation of nano-Co immune probes: Antibody 1 was rabbit anti-IgG monoclonal antibody against Listeria monocytogenes, antibody 2 was goat anti-rabbit IgG against Listeria monocytogenes. Buy nano-Co materials from the market, such as Beijing Deke Daojin Technology Co., Ltd. or Shanghai Chaowei Nanomaterials Co., Ltd., 20nm, purity 99.9%.

Silica-coated nano-Co: 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-Co 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 silicon is attached to Co.

Aminosilylated Co nanomaterials: The prepared silica-coated Co nanomaterials were 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 Co nanomaterials were vacuum-dried for 12 h.

Antibody modification: Take a certain amount of aminated Co 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, and resuspend. Due to the paramagnetic properties of nano-Co, when an external magnetic field is applied for separation, nano-Co 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. Pretreat 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. 1 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 there is target Listeria monocytogenes 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 plate prepared in step 2, then the probes combined with Listeria monocytogenes in the probe suspension will further combine with the enzyme plate The monoclonal antibody is 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 plate is the probes that bind L. monocytogenes.

4. Use the eluent (methanol, etc.) to elute the probe bound to Listeria monocytogenes on the microtiter 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, the spin-lattice relaxation time and spin-spin relaxation time measured in the solution are significantly different from the blank, indicating that there is a probe in the solution, thus indicating Listeria monocytogenes was present in the sample. The amount of probe is proportional to the spin-lattice relaxation time and the drop in spin-spin relaxation time. The greater the decrease, the more probes, which indirectly indicate the more Listeria monocytogenes, and the number of target bacteria in the sample can be quantitatively detected through standard addition verification. All food standards must not detect Listeria monocytogenes, and this method can quickly detect whether a sample contains Listeria monocytogenes.

detailed description

example 2

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

1. Nano-Co immunoprobe preparation: 1 antibody adopts O157:H7 rabbit anti-IgG monoclonal antibody, 2 antibody is O157:H7 goat anti-rabbit IgG, which can be monoclonal antibody or polyclonal antibody. Co nanoparticles can be purchased from the market (such as Chaowei Nanomaterials in Shanghai).

Silica-coated nano-Co: 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-Co 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 silicon is attached to Co.

Aminosilylated Co nanomaterials: The prepared silica-coated Co nanomaterials were 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 Co nanomaterials were vacuum-dried for 12 h.

Antibody modification: Take a certain amount of aminated Co nanoparticles, add excess 2 antibody, that is, goat anti-rabbit IgG antibody of Escherichia coli O157:H7, 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-Co, when an external magnetic field is applied for separation, nano-Co 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 1 antibody, that is, rabbit anti-IgG monoclonal antibody, that is, 100 µL of 100 µg/mL monoclonal antibody was 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. Pretreat 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 1 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 there is target Listeria monocytogenes 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 microplate prepared in step 2, then the probe combined with O157:H7 in the probe suspension will further combine with the monoclonal on the microplate. 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 O157:H7. All that remains on the plate is the probe bound to O157:H7.

4. Use the eluent (methanol, etc.) to elute the probe bound to E. coli O157:H7 on the microtiter 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, measure T1 and T2 of solution with nuclear magnetic resonance instrument (NMR20, New Mai company or miniNMR East China Normal University), take deionized water as blank, the spin-lattice relaxation time and spin-lattice relaxation time that solution records Compared with the blank, the spin relaxation time has a significant difference, indicating that there is a probe in the solution, thus indicating that there is Escherichia coli O157:H7 in the sample. The amount of the probe is related to the spin-lattice relaxation time and the spin-self proportional to the decrease in spin relaxation time. The greater the decrease, the more probes, which indirectly indicates the more Escherichia coli O157:H7, and the number of target bacteria in the sample can be quantitatively detected through standard addition verification.

Claims (3)

1., based on a NMR food-borne pathogen rapid detection for indirect enrichment of paramagnetic nano-Co probe, its characterization step is as follows:

1) add the specificity 1 detecting object bacteria in the sample to which to resist, if there is object bacteria in sample, then form 1 anti-compound by the combination of antibody antigen;

2) detect 1 antibody resisted of object bacteria, namely 2 anti-bags are by the preparation of paramagnetic nano Co probe;

3) what object bacteria specificity 1 resisted in ELISA Plate is fixing for subsequent use;

4) enrich target bacterium, and be separated: the 1st) sample of step process, add the 3rd) the obtained paramagnetic nano Co probe of step, abundant mixing concussion reaction a period of time, capture the after-applied externally-applied magnetic field of object bacteria, because nano Co has paramagnetic properties, Co probe can gather magnetic field, siphons away supernatant and then isolates probe; If have object bacteria in measuring samples, then first can the 1st) step time and the anti-compound of 1 anti-formation 1,1 anti-compound can again with 2 anti-compounds of detecting probe surface, by externally-applied magnetic field by enrichment, separation; The paramagnetic nano Co probe suspension that deionized water aseptic on a small quantity then forms object bacteria is added after washing, Magneto separate; Now, to catch and the Co probe of not catching 1 anti-compound still mixes;

5) the Co probe suspension of enrichment being added to the 3rd) step secures in the ELISA Plate of monoclonal antibody, if there is object bacteria, form double antibodies sandwich, by aseptic washed with de-ionized water, then the probe not grabbing object bacteria is just washed off, if there is not object bacteria, then all probes are all washed off;

6) after, wash with the probe of eluant, eluent by the double antibodies sandwich in ELISA Plate, drop off son and solvent by the method for externally-applied magnetic field separate probe by aseptic washed with de-ionized water, if also there is probe is exactly the probe catching object bacteria;

7) the 6th) probe of step gained, adds the suspension that aseptic deionized water forms probe, carries out NMR (Nuclear Magnetic Resonance) relaxation timing; Under fixing field intensity, the spin-lattice relaxation time of aseptic deionized water and spin spin relaxation time are steady state values, with aseptic deionized water for blank, the relaxation time spin-lattice relaxation time of the suspension recorded compares aseptic deionized water with spin spin relaxation time have remarkable reduction, then illustrate containing probe, thus have object bacteria in indirect proof sample, the decline of the amount of probe and spin-lattice relaxation time and spin spin relaxation time is proportional, by adding scalar quantity probe and indirect quantification goes out the amount of object bacteria.

2. the NMR food-borne pathogen rapid detection based on indirect enrichment of paramagnetic nano-Co probe according to claim 1, what it is characterized in that described object bacteria finally detects the change of evaluation method based on the relaxation time of nuclear magnetic resonance technique.

3. the NMR food-borne pathogen rapid detection based on indirect enrichment of paramagnetic nano-Co probe according to claim 1, is characterized in that described 1 resists specific monoclonal antibody for detecting object bacteria; 2 to resist be 1 anti-antibody.