CN110988349A - Two-channel detection method of capture probe and Escherichia coli O157: H7 and application thereof - Google Patents
Two-channel detection method of capture probe and Escherichia coli O157: H7 and application thereof Download PDFInfo
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Abstract
The invention discloses a capture probe, a double-channel detection method of Escherichia coli O157: H7 and application thereof, wherein the capture probe is Fe3O4(ii)/CuS, said Fe3O4the/CuS includes first preparing Fe3O4Micro-spheres and then mixing Fe3O4Adding microsphere into CTAB solution, adopting chemical deposition method, adding CuSO4With Na2S solution is stirred and washed to prepare Fe3O4and/CuS. The invention only needs to incubate bacteria and the capture probe, and avoids a complex labeling process. Solves the difficulty of antibody matching, and is simpler, more convenient and novel. Using Fe3O4the/CuS nano material has photo-thermal characteristics, and on the basis of naked eye detection, the photo-thermal detection mode improves the sensitivity detection limit by 10 times. The monoclonal antibody prepared by immunizing with the bacterial flagellin can only recognize Escherichia coli O157: H7 with high specificity, and has no specificity to other phyla bacteria. Can detect Escherichia coli O157: H7 in beef, chicken, milk, honey and other practical samples, has good application prospect, and can be used as a universal detection method for detecting all pathogenic bacteria.
Description
Technical Field
The invention belongs to the field of biological detection, and particularly relates to a dual-channel detection method of a capture probe and Escherichia coli O157: H7 and application thereof.
Background
Escherichia coli O157H 7 is a common serotype isolated from enterohemorrhagic Escherichia coli infection in humans, which is also the third most common bacterial food-borne pathogen after Salmonella and Campylobacter. Cattle and their meat products are considered to be the major source of E.coli O157 worldwide, and in addition, they are isolated from other animal meat products, such as chicken, pork, and lamb. After the Escherichia coli O157: H7 causes food infection and food poisoning, severe cramping abdominal pain and recurrent hemorrhagic diarrhea can occur, and meanwhile, the symptoms such as fever and vomiting are accompanied, so the Escherichia coli can be detected quickly, accurately, sensitively and simply, the method has important significance in the aspects of medical health, food health, animal epidemic disease monitoring and the like, and the method which is easy to operate, quick, portable and low in cost is still a huge technical challenge in order to detect the Escherichia coli O157: H7 more quickly and sensitively.
In recent years, immunochromatographic test strips have attracted extensive attention due to the advantages of convenient preparation and operation, low cost, short detection time, intuitive and reliable results and the like, and become mature rapid detection tools. In the aspect of improving the immunochromatographic test strip by the nano material, three obstacles still exist: (1) the single-reading detection form is easily interfered by the outside, and the detection sensitivity and accuracy are limited; (2) traditional materials, while advantageous, are limited to complex cross-linking between the material and the antibody; (3) antibody pairing is a very big obstacle, and it is difficult to obtain an antibody recognizing an antigen and a paired antibody in actual detection.
Disclosure of Invention
Aiming at the defects and shortcomings in the prior art, in order to solve the technical problems that a single-reading detection mode is easily interfered by the outside and the sensitivity and the accuracy are limited, the invention adopts a dual-channel detection method of a capture probe and Escherichia coli O157: H7 and application thereof.
In order to achieve the purpose, the technical scheme adopted by the invention comprises the following steps:
a capture probe, the capture probe is Fe3O4(ii)/CuS, said Fe3O4the/CuS includes first preparing Fe3O4Micro-spheres and then mixing Fe3O4Adding microsphere into CTAB solution, adopting chemical deposition method, adding CuSO4With Na2S solution is stirred and washed to prepare Fe3O4/CuS。
Further, said Fe3O4The addition amount of the microspheres is 0.1-0.3 g, and the particle size is 220-280 nm.
Furthermore, the particle size of the CuS is 240-420 nm.
Preferably, Fe is added3O4Adding the microspheres into CTAB solution, stirring, and suspending in Na2Adding CuSO into the S aqueous solution dropwise4Continuously stirring the aqueous solution, washing and drying to obtain the water-soluble organic fertilizer.
A dual-channel detection method of Escherichia coli O157: H7 comprises the steps of capturing Escherichia coli O157: H7 in a sample to be detected by using the capture probe, incubating to obtain a liquid to be detected, dripping the liquid to be detected onto a test strip for detecting Escherichia coli O157: H7, and irradiating the test strip by adopting near infrared light to realize dual-channel detection.
Further, the concentration of the capture probe is 0.8-1.2 mg/mL, the incubation time is 10-900 s, and the wavelength of the near-infrared light is 808 nm.
Further, the test strip comprises a lining plate, a nitrocellulose membrane is attached to the lining plate, one end of the nitrocellulose membrane covers a water absorption pad, the other end of the nitrocellulose membrane sequentially covers a sample pad and a combination pad, and a detection line is transversely arranged on the non-covered surface of the nitrocellulose membrane; the detection line is coated with Escherichia coli O157: H7 monoclonal antibody, the binding pad and the sample pad are respectively sealed by sealing liquid, and the test strip has no quality control line.
Specifically, the preparation method of the detection line coated with the Escherichia coli O157: H7 monoclonal antibody comprises the following steps: dissolving an Escherichia coli O157H 7 monoclonal antibody in a coating solution to prepare an antibody coating solution of 1mg/mL, and coating the antibody coating solution on a detection line away from a nitrocellulose membrane at the speed of 1 mu L/cm;
the coating liquid is as follows: 0.02g of sodium azide, 0.8g of sodium chloride, 0.29g of disodium hydrogen phosphate dodecahydrate, 0.02g of potassium chloride and 0.02g of potassium dihydrogen phosphate are added with water to reach the constant volume of 100mL, and the sodium azide-potassium dihydrogen phosphate-potassium hydrogen phosphate-potassium dihydrogen phosphate-potassium hydrogen phosphate-.
Specifically, the detection limit of the test strip on Escherichia coli O157H 7 is 102CFU/mL。
The capture probe is used for detecting Escherichia coli O157: H7 in beef, chicken, milk and honey.
The dual-channel detection method of Escherichia coli O157: H7 is used for detecting Escherichia coli O157: H7 in beef, chicken, milk and honey.
Compared with the prior art, its advantage lies in with positive effect:
(1) and (4) label exemption. Only bacteria need to be incubated with the capture probe, avoiding the complex labeling process.
(2) Breaks through the traditional sandwich detection method. The invention only uses one antibody to scratch on the nitrocellulose membrane for direct detection, breaks through the traditional sandwich detection method which simultaneously adopts two antibodies, greatly saves the cost, solves the difficulty of antibody matching, and is simpler, more convenient and novel.
(3) And (4) double-channel detection. The invention utilizes Fe3O4the/CuS nano material has a photo-thermal characteristic, and on the basis of naked eye detection, the photo-thermal detection mode improves the sensitivity detection limit by 10 times.
(4) The sensitivity is high. The test strip provided by the invention has the lowest detection limit of 10 to Escherichia coli O157H 72CFU/mL, the value is lower than that reported in many other documents.
(5) The specificity is high. The monoclonal antibody prepared by immunizing with the bacterial flagellin can only recognize Escherichia coli O157: H7 with high specificity, and has no specificity to other phyla bacteria.
(6) Good practical application. The kit can detect Escherichia coli O157: H7 in actual samples such as beef, chicken, milk, honey and the like, has good application prospect, and can be used as a universal detection method for detecting all pathogenic bacteria.
Drawings
FIG. 1 is an assembly diagram of an immunochromatographic strip for detecting Escherichia coli O157: H7 according to the present invention;
FIG. 2 is a schematic flow chart of immunochromatography for rapidly detecting Escherichia coli O157: H7 in accordance with the present invention;
FIG. 3 shows the optimized result of the immunochromatographic test strip prepared by the present invention;
FIG. 4 shows the detection sensitivity of the immunochromatographic test strip prepared in the present invention;
FIG. 5 shows the specificity of the immunochromatographic test strip prepared in the present invention;
FIG. 6 shows the practical application of the immunochromatographic test strip prepared in the present invention;
FIGS. 7 and 8 are verifications of novel probes made according to the present invention;
the following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
Detailed Description
Compared with a single-reading analysis strategy, the double-reading rapid detection immunochromatography has higher accuracy and higher sensitivity, and recently discovered photothermal effect is used for enhancing the sensitivity of an immunochromatography test strip and photothermal materials, and the results of the photothermal nanomaterials enable current researches to propose photothermal immunoassay and construct a plurality of small-scale assembled immune biosensors.
Compared with the traditional method, the preparation method provides a dual-mode immunochromatographic test strip detection method based on the unmarked and photothermal effects, and is used for detecting Escherichia coli O157: H7. The invention relates to a double-channel detection method, which comprises the following steps: in one aspect, Fe3O4the/CuS can effectively capture bacteria and does not need the step of labeling the antibody in the traditional sandwich modeThis novel label-free immunochromatographic test strip detection strategy uses only one antibody, and bacteria serve as an intermediate for linking materials and antibodies together, saving costs and achieving high sensitivity detection. On the other hand, based on the photo-thermal effect of the material, a simple sensor device is manufactured by using a 808nm near-infrared light laser pen, an infrared camera and a smart phone, near-infrared light irradiates the material accumulated on the T line, the temperature difference (delta T) before and after the irradiation of the T line of each test strip is recorded, and the accuracy and the sensitivity of the biosensor are further improved. Coli O157H 7 in the sample was first Fe3O4the/CuS capture, after which the material that has adsorbed the bacteria will be captured by the monoclonal antibody immobilized thereon, on the T-line, a visibly apparent brown color will appear. In addition, due to the accumulation of the nanomaterial having photothermal properties on the T-line, the temperature of the material increases under laser irradiation, and therefore, Fe3O4The brown and photothermal signals of/CuS are used as quantitative readings in the immunochromatographic test strip, and a new way is provided for detecting various food-borne pathogens by using bacteria and nano materials which are directly combined as a strong probe. "Fe" of the invention3O4the/CuS' refers to a rhombic cluster-shaped material CuS and a spherical magnetic nano material Fe which are prepared by the preparation method3O4And compounding.
The working principle of the invention is as follows: based on Fe3O4the/CuS nano material can directly adsorb bacteria, and Fe is added during detection3O4the/CuS was added directly to a bacterial suspension of E.coli O157: H7, which was immediately adsorbed on the bacterial surface. Then, the novel probe complex is added on a sample pad, and the sample pad is absorbed by the sample pad under the traction of capillary force, and the complex is captured by the immobilized Escherichia coli O157: H7 monoclonal antibody on the T line due to the specific binding between the monoclonal antibody and Escherichia coli O157: H7 antigen. At this time, the surface of the bacteria is enriched with a large number of nano-material probes, and the probes are deposited on the T line to play a role in visual detection. In addition, in the detection process of the photothermal immunosensor on Escherichia coli O157: H7, near infrared light of 808nm is used for irradiating T rays and passes through a photothermal converterAnd measuring the change of the temperature, and as the concentration of the compound increases, more nano materials combined with the bacteria are accumulated on the T line, and the change of the temperature is more obvious, so that the photothermal detection of the bacteria is realized.
Through the construction, Escherichia coli O157: H7 can be effectively captured by the antibody on the T line, the traditional mode that a sandwich test strip for testing the antibody and capturing the antibody is adopted is broken through, only one antibody is used, the cost is greatly reduced, and highly sensitive detection is realized. In addition, the photothermal effect test strip is developed to solve the problem of low sensitivity detection limit at present, and the test strip detection method has the advantages of high sensitivity, strong specificity, simple structure, high cost benefit, quick analysis time and portability, and great application potential to meet the requirement of instant diagnosis and determination. The method is successfully applied to the detection of salmonella in beef, chicken, milk and honey, and the practicability and the applicability of the method are verified. Therefore, only one antibody is needed, and the method can be used as a universal detection platform to detect all pathogenic bacteria.
The reagents used in the invention are all obtained in the market, and the instruments are all conventional instruments. The chemical deposition method is a chemical reduction process in which metal ions in a plating solution are reduced using a suitable reducing agent and deposited on the surface of a substrate.
The E.coli O157: H7 monoclonal antibody may be one prepared by a conventional method or one commercially available, and is preferably one prepared according to the method described in Zhang Daohong et al, publication of Analytical Chimica Acta, vol.635, entitled "Production of an ultra-reactive genetic monoclonal antibodies using a modified two-step screening procedure": the method comprises the steps of immunizing a mouse with Escherichia coli O157: H7 to induce the generation of antiserum for resisting Escherichia coli, then taking splenocytes of the mouse to perform semisolid cell fusion with SP2/0 myeloma cells, and screening hybridoma cells capable of generating a target antibody through indirect ELISA and sandwich ELISA to obtain 5 strains of Escherichia coli O157: H7 monoclonal antibodies with high sensitivity and specificity, wherein the two strains of antibodies can be paired to form a sandwich. And injecting the hybridoma cells with the best titer into the abdominal cavity of the mouse, generating a large amount of antibodies, and purifying the antibodies by adopting an ammonium caprylate method.
Example 1:
(1) FeCl is prepared by hydrothermal synthesis3·6H2O (1.35g) and trisodium citrate (0.45g) were mixed in ethylene glycol (40mL) and stirring continued for 60 minutes, NaAc (2.4g) was added to the mixture and stirred for 0.5 hours, then the mixture was sealed in a Teflon-lined stainless steel autoclave and heated at 200 ℃ for 10 hours, after cooling to room temperature, the product was washed three times with water and ethanol and dried in a 60 ℃ drying cabinet to give Fe3O4And (3) microspheres.
(2) Adopting a chemical deposition method to obtain Fe obtained in the step one3O4(0.2g) microspheres were added to CTAB solution (0.5g/L25mL cetyltrimethylammonium bromide), stirred for 0.5 h, the solution was washed three times, stirred at room temperature for 3 h, suspended in Na2To an aqueous S solution (0.04M, 25mL) was added CuSO dropwise4The aqueous solution (0.04M, 25mL) was stirred continuously for 3 hours, the mixed solution was darkened, washed several times with water and ethanol, and then dried at 60 ℃.
Prepared Fe3O4The solution to be detected is obtained by mixing and incubating 1mg/mL aqueous solution prepared from/CuS and 100 mu L solution of Escherichia coli O157: H7 for four minutes, and can be used for test strip detection.
The solutions to be tested used in examples 3 to 5 described below were prepared as in example 1.
Example 2:
the preparation method of the Escherichia coli O157H 7 universal monoclonal antibody comprises the following steps:
the method comprises the following steps: animal immunization
BALB/c mice of 6 weeks old were purchased, E.coli O157: H7 was purchased, and flagellin was extracted for immunization. For the first immunization, 0.8mg/mL of Escherichia coli O157H 7 flagellin solution and an equal amount of Freund's complete adjuvant are mixed and emulsified, and the emulsified antigen is injected into the abdominal cavity of a mouse; after 4 weeks, the second immunization was carried out, and 0.8mg/mL E.coli O157: H7 flagellin was mixed with an equal amount of Freund's incomplete adjuvant and emulsified antigen was injected into the abdominal cavity of mice. A third immunization was performed after 4 weeks in the same manner as the second immunization; a fourth immunization was performed three weeks later, in the same manner as the second immunization. The 4 times of immunization are the same, and 100 mug of Escherichia coli O157H 7 is adopted in each mouse; one week after the 4 immunizations, tail vein blood is collected, serum is separated, and the titer of the mouse serum is monitored by adopting an indirect ELISA method; and selecting a mouse corresponding to the serum with relatively high titer for the last boosting immunization, wherein the immunization dose is 2 times of that of the previous mouse, and the immunization mode is intraperitoneal injection.
The 0.8mg/mL escherichia coli O157H 7 flagellin solution is obtained by dissolving 0.8mg of extracted flagellin in 0.01mol/L phosphate buffer salt solution, wherein the phosphate buffer salt solution is 0.02g of sodium azide, 0.8g of sodium chloride, 0.29g of disodium hydrogen phosphate dodecahydrate, 0.02g of potassium chloride and 0.02g of potassium dihydrogen phosphate, and adding water to a constant volume of 100 mL.
Step two: cell fusion
3 days after the boosting immunization, the mice after the boosting immunization adopt 50 percent polyethylene glycol (with the molecular weight of 1450g/mol) as a fusion agent to carry out cell fusion according to a conventional method. The specific method comprises the following steps:
(1) take 1 to 2X 107Mixing SP2/0 myeloma cells with immune spleen cells, centrifuging at 800rpm for 7min, and washing the cells twice;
(2) taking out sterilized absorbent paper in an ultraclean workbench, pouring out supernatant of a 50mL centrifuge tube filled with myeloma cell and immune spleen cell mixed cells, and inversely buckling the centrifuge tube on the absorbent paper to remove obvious water drops;
(3) from CO2Taking 50% PEG which is incubated to 37 ℃ out of the incubator, sucking 0.8mL by using a 1mL suction tube, holding a 50mL centrifuge tube with mixed cells by hand, placing the centrifuge tube in a 37 ℃ water bath, slowly adding PEG to the mixed cells, gently stirring while adding, continuously adding for 90s, standing for 1min, moving a water bath pot, taking out 50mL RPMI-1640 basic culture solution which is incubated to 37 ℃, sucking 10mL by using the suction tube, slowly adding to the fused cells, gently stirring while adding to disperse cell blocks, firstly adding 1mL, then adding 2mL, then adding 3mL, finally adding the rest 4mL, adding the first 10mL, then adding the rest 40mL along the tube wall, screwing a cover, and slightly reversing for several times to mix the cells;
(4) centrifuging at 10000rpm for 7min, discarding the supernatant, and resuspending the fused cells with 20mL HAT complete culture solution with the suspended feeder cells;
(5) adding the resuspended cells into 80mL of semisolid culture medium incubated to 37 ℃, gently shaking to mix the cells uniformly, then sucking up the semisolid culture medium suspended with the fused cells by using a 20mL syringe, uniformly distributing the semisolid culture medium into 8-9 six-well cell culture plates at 1.5-2 mL/well, and placing the plates in a 37 ℃ CO solution2Culturing in an incubator;
(6) observing about 2-3 weeks after fusion, observing that white cell colonies appear on the semisolid culture medium, sucking single colonies one by using a sterile gun head, and transferring the single colonies into a 96-hole culture plate, wherein one clone is cloned in each hole. The culture was continued with HT liquid medium. When the clone grows to the full-length hole bottom 1/2-2/3, the culture solution turns yellow, and then the screening of hybridoma cells can be carried out.
Step three: screening of cell lines
Screening out positive holes for resisting Escherichia coli O157: H7 by adopting an indirect ELISA method; and then, carrying out pairing detection on the screened positive holes by adopting a sandwich ELISA method, taking Escherichia coli O157: H7 as an antigen, and selecting holes with higher light absorption values and sensitivity. Obtaining a monoclonal cell strain capable of pairing.
Step four: purification of monoclonal antibodies
Among the screened cell lines, 2B4 cell line produced antibody with the best sensitivity to Escherichia coli O157: H7, so 2B4 cell line was selected and injected into BALB/c mice treated with Freund's incomplete adjuvant in advance, ascites of the mice were collected, and the antibody was purified by the caprylic acid-ammonium sulfate method, which was specifically performed as follows: the ascites fluid was filtered with double-layer filter paper, centrifuged at 12000r/min at 4 ℃ for 15min, and the supernatant was aspirated. Mixing the obtained ascites supernatant with 2 times volume of acetate buffer solution, slowly adding 33 mu L/mL of ascites of n-octanoic acid under stirring, mixing at room temperature for 30min, and standing at 4 ℃ for 2 h. Centrifuging at 12000r/min for 30min at 4 deg.C, and discarding the precipitate. After the supernatant was filtered through a 0.22 μm filter, 1/10 volumes of 0.1mol/L phosphate buffer solution with pH7.4 were added, and the pH of the mixture was adjusted to 7.4 with 2mol/L sodium hydroxide solution. Precooling the supernatant at 4 ℃, slowly adding an equal volume of saturated ammonium sulfate solution, and standing for 2h at 4 ℃. Centrifuging at 12000r/min at 4 deg.C for 30min, and discarding the supernatant. The obtained precipitate is resuspended in 1/10 phosphate buffer solution with volume of original ascites volume of 0.02mol/L, and is put into a dialysis bag, and is dialyzed for 6-8h by 0.01mol/L phosphate buffer solution, and then is dialyzed by pure water. After dialysis, the liquid was taken out at 4 ℃ and centrifuged at 12000r/min for 30min, and the precipitate was discarded. And (3) freezing the fully dialyzed protein solution in a refrigerator at the temperature of-80 ℃, then freeze-drying the protein solution by using a freeze dryer, collecting the freeze-dried powder to obtain a purified Escherichia coli O157: H7 monoclonal antibody, and placing the antibody in a refrigerator at the temperature of-20 ℃ for later use.
The acetate buffer solution is 0.29g of sodium acetate, and 0.141mL of acetic acid is added with water and is metered to 100 mL.
The 0.01mol/L phosphate buffer solution is obtained by adding 0.8g of sodium chloride, 0.29g of disodium hydrogen phosphate dodecahydrate, 0.02g of potassium chloride and 0.02g of monopotassium phosphate into water to fix the volume to 100 mL. The sodium hydroxide solution of 2mol/L is obtained by adding 80g of sodium hydroxide and water to a constant volume of 1000 mL.
Example 3: this example presents a condition-optimized experiment for a test strip for rapid detection of E.coli O157: H7.
(1) Preparation of nitrocellulose membranes
Coating of detection lines: dissolving the Escherichia coli O157H 7 monoclonal antibody in the coating solution to prepare a solution of 1 mg/mL; the coating solution was applied laterally to the nitrocellulose membrane at a position 30mm away from the surface (i.e., on the detection line) at a speed of 1. mu.L/cm by streaking, and then dried at 37 ℃ for 30 minutes. The coating liquid is as follows: 0.02g of sodium azide, 0.8g of sodium chloride, 0.29g of disodium hydrogen phosphate dodecahydrate, 0.02g of potassium chloride and 0.02g of potassium dihydrogen phosphate, and water is added to the mixture to reach a constant volume of 100 mL.
(2) Preparation of sample pad: cutting the glass fiber membrane into pieces with length of 15mm and width of 3mm, soaking in sealing solution, drying at 37 deg.C for 10-16 hr to obtain sample pad, and storing in a desiccator at room temperature. The confining liquid is prepared by adding water into 2g of bovine serum albumin, 0.02g of sodium azide, 0.8g of sodium chloride, 0.29g of disodium hydrogen phosphate dodecahydrate, 0.02g of potassium chloride and 0.02g of potassium dihydrogen phosphate to a constant volume of 100 mL.
(3) Preparation of the bonding pad: cutting the glass fiber membrane into pieces with length of 8mm and width of 3mm, soaking in sealing solution, taking out, drying at 37 deg.C for 10-16 hr, and storing at room temperature in a dryer.
(4) Preparation of absorbent pad
Cutting the absorbent paper into pieces with the length of 18mm and the width of 3mm to obtain the absorbent pad.
(5) The microbial culture comprises the following steps: activating Escherichia coli O157H 7, inoculating to LB culture medium, standing at 37 deg.C, and culturing for 24 hr; selecting a single colony, inoculating the single colony in 250mL LB broth culture medium, and culturing the single colony for 24 hours at 37 ℃ by a shaking table at 150 r/min; centrifuging the bacterial liquid at 4000r/min for 15min, and collecting thalli; respectively washing the thallus 3 times with 0.01mol/L phosphate buffer solution with pH7.4 concentration, and suspending with 10mL0.01 mol/L phosphate buffer solution; adding 0.5% formalin solution, and standing at room temperature for 24 hr for inactivation; after inactivation, washing with 0.01mol/L phosphate buffer solution for 3 times, adjusting to appropriate concentration with 0.01mol/L PBS, and adjusting antigen concentration to 108CFU/mL, stored at-20 ℃ for further use.
(6) Assembling the test strip: firstly, attaching the nitrocellulose membrane to a lining plate, then pressing the sample pad by 1-3mm, pressing the nitrocellulose membrane by 1-3mm, and pressing the nitrocellulose membrane by 1-3mm through a water absorption pad to be sequentially attached to the lining plate, thus obtaining the immunochromatography test strip for rapidly detecting Escherichia coli O157: H7.
(7)Fe3O4Optimization of/CuS solution concentration
Fe3O4The concentration of/CuS has a significant influence on the coloring effect of the bacteria. At a fixed bacterial concentration of 108Different Fe were studied under CFU/mL3O4The concentration of/CuS was 0.5, 1.0, 1.5 and 2.0mg/mL, respectively.
(8)Fe3O4Optimization of/CuS solution volume
Fe3O4The volume of/CuS plays an important role in the detection capability of the method, and the volume quantity influences the detection sensitivity. In this step, different dye volumes of 10 to 80. mu.L, respectivelyAnd 100. mu.L of 106CFU/mL bacterial solution mixed incubation and test.
(9) Time of immune response
Another important parameter that affects the intensity of the test line is the immune response time. The result was measured every 5 minutes for 5-15 minutes after the stained bacteria were added to the sample pad.
(10)Fe3O4Optimization of incubation time of CuS solution with bacterial solution
The time of incubation may also affect the signal intensity on the detection line. Mixing Fe3O4the/CuS and bacteria mixture was incubated for different times in the range of 10, 60, 120, 180, 240, 360, 600, 900 s.
The results are shown in FIG. 3A, with Fe3O4And the color intensity of the detection line is in an increasing trend due to the increase of the concentration of/CuS. When Fe3O4The intensity of the T line is the highest when the concentration of the/CuS is as high as 1.0mg/mL, and the concentration is the optimal concentration required by the experiment.
See FIG. 3B, with Fe3O4Volume increase of/CuS, volume increased to 40. mu.L, reaching maximum. Thus, Fe3O4The optimal volume of/CuS is 40. mu.L.
As shown in FIG. 3C, the intensity of the test line was maximal at 10min as the immunization time was extended. Therefore, the reaction time is set to 10 minutes to save the measurement time and improve the readability and accuracy of the visualization effect.
As shown in FIG. 3D, the color intensity on the test line with increasing incubation time was greatest at 4 min. Thus, in the subsequent experiments, Fe3O4the/CuS and bacteria were incubated for 4 minutes and then added to the sample pad.
Example 4:
sensitivity determination of test strip for rapidly detecting Escherichia coli O157H 7
The test strip preparation and bacterial culture process steps are the same as the steps (1) to (6) in example 3.
The specific detection process comprises the following steps: mixing Fe3O4Dissolving CuS in water to obtain 1.0mg/mL solution, and adding 0.01M phosphate buffer solutionDiluting to 80-10%8CFU/mL concentration, 100. mu.L solution was taken as the detection solution for each concentration, and 40. mu.L Fe3O4the/CuS mixture was incubated for 4 minutes, and then the sample pad of the test strip was added dropwise, while taking 100. mu. L0.01M phosphate buffer as a negative control, the same procedure as above was followed, and the sample pad of another test strip was added dropwise, and the results were read after 10 minutes.
And (3) visual detection results: (1) positive: when the detection line of the detection test strip shows a brown line, the result is positive, and the concentration of Escherichia coli O157H 7 in the sample to be detected is higher than or equal to 103CFU/mL. (2) Negative: when the detection line of the detection test strip does not display color, the result is negative, which indicates that the Escherichia coli O157H 7 in the sample to be detected is less than 103CFU/mL。
As shown in FIG. 4A, the test strip T line has lighter brown color and a macroscopic concentration of 10 as the concentration of Escherichia coli O157: H7 is reduced3CFU/mL, therefore, the lowest concentration of Escherichia coli O157: H7 detectable by naked eyes is 103CFU/mL。
As shown in FIG. 4B, as the concentration of Escherichia coli O157: H7 is reduced, the detected photothermal temperature is lower, and the detected concentration of the photothermal test strip is 102CFU/mL, compared with the meat eye detection sensitivity improved by 10 times.
Example 5: specificity determination of test strip for rapidly detecting salmonella enteritidis
The test strip preparation and bacterial culture process steps were the same as in (1) to (6) of example 2.
The specific detection process comprises the following steps: mixing Fe3O4Dissolving CuS in water to obtain 1.0mg/mL solution, and diluting the bacterial solution of Escherichia coli O157H 7, Salmonella typhimurium, Salmonella hadamara, Salmonella london, Staphylococcus aureus, Campylobacter jejuni, Listeria monocytogenes and Salmonella enteritidis to 10 with 0.01M phosphate buffer solution8CFU/mL concentration, 100. mu.L solution was taken as the detection solution for each concentration, and 40. mu.L Fe3O4the/CuS mixture is incubated for 4 minutes, and then the sample pad of the test strip is added dropwise, and 100 mu L0.01M phosphate buffer is takenThe wash was used as a negative control and the procedure was the same as above, and the sample pad of another test strip was added dropwise and the results were read after 10 minutes.
And (3) visual detection results: (1) positive: and when the detection line of the detection test strip shows a brown line, judging the test strip to be positive. (2) Negative: and when the detection line of the detection test strip does not display the color, judging as a negative result.
Referring to FIG. 5, numerals 1-9 each represent 108CFU/mL of different strains are sequentially Escherichia coli O157: H7, Salmonella typhimurium, Salmonella hadamara, Salmonella london, Staphylococcus aureus, Campylobacter jejuni, Listeria monocytogenes and Salmonella enteritidis. The test strip T line for detecting other bacteria has no color except that the test strip T line for detecting the Escherichia coli O157: H7 has bright brown color which is visible to naked eyes, which indicates that the invention can highly specifically recognize the Escherichia coli O157: H7 and has particularly high specificity.
Example 6: application of test strip for rapidly detecting Escherichia coli O157H 7
The test strip preparation and bacterial culture process steps were the same as in (1) to (6) of example 2.
The specific detection process comprises the following steps: mixing Fe3O4Dissolving CuS in water to obtain solution with concentration of 1.0mg/mL, adding bacterial solution with known concentration into drinking water to form 80-108CFU/mL concentration, 100. mu.L solution was taken as the detection solution for each concentration, and 40. mu.L Fe3O4And performing mixed incubation on CuS for 4 minutes, dropwise adding the sample pad of the test strip, simultaneously taking 100 mu L of drinking water as negative control solution, dropwise adding the sample pad of another test strip in the same operation as the above, reading the result after 10 minutes, and irradiating the T ray by using near infrared light of 808nm to obtain the temperature difference.
And (3) visual detection results: (1) positive: and when the detection line of the detection test strip shows a brown line, judging the test strip to be positive. (2) Negative: and when the detection line of the detection test strip does not display the color, judging as a negative result.
As shown in FIG. 6, as the concentration of bacteria decreases, the brown color on the test strip becomes lighter and lighter, and the actual samples of beef, chicken, milk and honey are visually detectedThe out-limit concentration can reach 10 respectively4，103，104，103CFU/mL, photothermal detection limit concentration of 103，102，103，102CFU/mL reflects the good practical application value.
Example 7, Fe3O4Characterization of the/CuS Material
To demonstrate the use of Fe in the present invention3O4The inventors also made the following experiments:
(1) scanning electron microscope: FIG. 7A is Fe3O4A scanning electron microscope image is spherical, and the particle size is 220-280 nm; FIG. 7B is Fe3O4Electron microscope picture of/CuS composite nano material, diamond-shaped cluster-shaped material CuS (240-420 nm) and spherical magnetic nano material Fe3O4The composite material is formed; FIG. 7C is Fe3O4The graph of/CuS capturing bacteria shows that a plurality of composite nano materials are attached to the surface of the bacteria, and the bacteria are broken and incomplete, which indicates that Fe3O4the/CuS composite nanomaterial can capture and kill bacteria, and fig. 7D insets a and b show that, a diagram is a bacterial solution with OD600 ═ 1 in the absence of any external magnetic field, and b diagram is a bacterial solution with OD600 ═ 1 and Fe3O4The mixed solution of/CuS, beside which a magnet was placed, was prepared by mixing a bacterial solution with Fe at an OD600 of 1.0 without any external magnetic field3O4After the/CuS mixture, the mixture was almost transparent within 30 minutes after the magnet was applied.
(2) Bacteriostatic map: 7E is Fe3O4The antibacterial tests of/CuS and three bacteria, salmonella enteritidis, Escherichia coli O157: H7 and Listeria are used in test detection, and the number of bacteria in experimental groups (a, b and c) is obviously less than that in control groups (d, e and f), which indicates that the capture probe has strong antibacterial ability.
(3) Ultraviolet characterization: as can be seen from FIG. 7F, it is similar to the original Fe3O4In contrast, nanocomposite Fe3O4The characteristic peak of/CuS (dotted line) is deep red shifted 570 nm.
(4) Infrared characterization: from the figure8A indicates that Fe3O4the/CuS nano composite material not only has the characteristic Cu-S peak (1110 cm) of CuS-1) And Fe3O4Fe-O peak of (594 cm)-1)，1637cm-1And 1401cm-1The band at (b) is attributable to tensile vibration of COOH.
(5) X-ray photoelectron spectroscopy: as can be seen from fig. 8B, at 934.16, 530.41, 168.79, 284.94 and 711.34eV, there are five different peaks on the curve for Cu 2p, O1S, S2 p, C1S and Fe 2p, respectively, indicating successful material synthesis.
(6) X-ray diffraction: as can be seen from FIG. 8C, Fe3O4the/CuS nanocomposite has characteristic peaks of CuS of 29.08 ° (102),31.54 ° (103),34.57 ° (006),49.56 ° (110) and 57.98 ° (116), and also has Fe3O4The characteristic peaks of (1) 32.40 DEG (220),36.57 DEG (331), 43.20 DEG (400), 54.87 DEG (422),56.30 DEG (511) and 62.17 DEG (440), which proves the success of the synthesis of the composite material.
(7) And (3) photothermal characterization: from FIG. 8D, following Fe3O4The temperature of the material rises rapidly due to the increase of the irradiation time and concentration of the/CuS, and the material is kept unchanged after about 3 minutes, however, a 1ml pure water sample does not show any obvious temperature change after irradiation, and the result shows that the nano material has good photothermal effect and can effectively convert light energy into heat energy.
Claims (10)
1. A capture probe, characterized in that the capture probe is Fe3O4(ii)/CuS, said Fe3O4the/CuS includes first preparing Fe3O4Micro-spheres and then mixing Fe3O4Adding microsphere into CTAB solution, adopting chemical deposition method, adding CuSO4With Na2S solution is stirred and washed to prepare Fe3O4/CuS。
2. The capture probe of claim 1, wherein the Fe is3O4The addition amount of the microspheres is 0.1-0.3 g, and the particle size is 220-280 nm.
3. The capture probe of claim 1 or 2, wherein the CuS has a particle size of 240 to 420 nm.
4. A dual-channel detection method of Escherichia coli O157H 7 is characterized by comprising the steps of capturing Escherichia coli O157H 7 in a sample to be detected by using the capture probe of any claim of claims 1-3, incubating to obtain a liquid to be detected, dripping the liquid to be detected onto a test strip for detecting Escherichia coli O157H 7, and irradiating the test strip by adopting near infrared light to realize dual-channel detection.
5. The dual-channel detection method of Escherichia coli O157H 7 as claimed in claim 4, wherein the concentration of the capture probe is 0.8-1.2 mg/mL, the incubation time is 10-900 s, and the wavelength of the near infrared light is 808 nm.
6. The dual-channel detection method of Escherichia coli O157H 7 as claimed in claim 4, wherein the test strip comprises a lining plate, a nitrocellulose membrane is attached on the lining plate, one end of the nitrocellulose membrane covers a water absorption pad, the other end of the nitrocellulose membrane sequentially covers a sample pad and a binding pad, and a detection line is transversely arranged on the non-covered surface of the nitrocellulose membrane; the detection line is coated with Escherichia coli O157: H7 monoclonal antibody, the binding pad and the sample pad are respectively sealed by sealing liquid, and the test strip has no quality control line.
7. The dual-channel detection method of Escherichia coli O157: H7 as claimed in claim 6, wherein the detection line coated with Escherichia coli O157: H7 monoclonal antibody comprises: dissolving an Escherichia coli O157H 7 monoclonal antibody in a coating solution to prepare an antibody coating solution of 1mg/mL, and coating the antibody coating solution on a detection line away from a nitrocellulose membrane at the speed of 1 mu L/cm;
the coating liquid is as follows: 0.02g of sodium azide, 0.8g of sodium chloride, 0.29g of disodium hydrogen phosphate dodecahydrate, 0.02g of potassium chloride and 0.02g of potassium dihydrogen phosphate are added with water to reach the constant volume of 100mL, and the sodium azide-potassium dihydrogen phosphate-potassium hydrogen phosphate-potassium dihydrogen phosphate-potassium hydrogen phosphate-.
8. The dual-channel detection method for detecting Escherichia coli O157H 7 as claimed in claim 6, wherein the detection limit of the test strip for Escherichia coli O157H 7 is 102CFU/mL。
9. Use of the capture probe of any of claims 1-4 for detecting E.coli O157: H7 in beef, chicken, milk and honey.
10. The dual-channel detection method of Escherichia coli O157: H7 as claimed in any one of claims 5-8 is used for detecting Escherichia coli O157: H7 in beef, chicken, milk and honey.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111948397A (en) * | 2020-06-09 | 2020-11-17 | 江苏海洋大学 | Double-antibody sandwich ELISA method for rapidly detecting Escherichia coli O157: H7 |
| CN114113585A (en) * | 2021-11-04 | 2022-03-01 | 西北农林科技大学 | Double-signal probe, test strip for detecting escherichia coli and application |
Citations (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007037282A1 (en) * | 2005-09-27 | 2007-04-05 | Eisai R & D Management Co., Ltd. | Method of forming autoaggregate on microparticle and method of detecting target analyte |
| WO2009026141A1 (en) * | 2007-08-17 | 2009-02-26 | University Of Florida Research Foundation, Inc. | Supercrystalline colloidal particles and method of production |
| CN103116026A (en) * | 2013-01-29 | 2013-05-22 | 南昌大学 | Quick detection method for food-borne pathogens based on immunomagnetic separation of Fe3O4 nano materials |
| CN103383397A (en) * | 2013-06-24 | 2013-11-06 | 西北农林科技大学 | Alicyclobacillus immunomagnetic microspheres and application thereof |
| CN103439496A (en) * | 2013-08-13 | 2013-12-11 | 南昌大学 | Escherichia coli O157:H7 enrichment and rapid detection method |
| CN104749365A (en) * | 2013-12-31 | 2015-07-01 | 中国科学院上海微系统与信息技术研究所 | Difunctional composite nanosphere and method for rapidly detecting food-borne pathogenic bacteria |
| CN105527428A (en) * | 2016-01-19 | 2016-04-27 | 南昌大学 | Method for fast detecting colon bacillus O157:H7 |
| CN105651991A (en) * | 2016-01-19 | 2016-06-08 | 南昌大学 | Method for rapidly detecting enterobacter sakazakii |
| US20160172027A1 (en) * | 2014-12-16 | 2016-06-16 | Conversant Intellectual Property Management Inc. | Polymer Memory |
| CN106290882A (en) * | 2015-06-01 | 2017-01-04 | 上海凯创生物技术有限公司 | A kind of Candida albicans antigen near-infrared fluorescent detection kit and application thereof |
| CN106290320A (en) * | 2016-09-09 | 2017-01-04 | 南昌大学 | A kind of OTA chemical luminescence detection method based on unmarked aptamer sensor |
| CN107389919A (en) * | 2017-07-27 | 2017-11-24 | 广东省微生物研究所（广东省微生物分析检测中心） | A kind of label-free fluorescence aptamer sensor and its preparation method and application |
| CN108020674A (en) * | 2017-12-22 | 2018-05-11 | 天津现代职业技术学院 | It is a kind of to utilize the attached quick method for differentiating strain level pathogenic bacteria in food of double suction |
| CN108303532A (en) * | 2017-12-15 | 2018-07-20 | 浙江工商大学 | A kind of escherichia coli O157 of non-diagnostic purpose:The rapid detection method of H7 |
| CN108715889A (en) * | 2018-04-27 | 2018-10-30 | 曾小敏 | A kind of state enterorrhagia Bacillus coil 0157 living:The rapid detection method of H7 |
| CN109738638A (en) * | 2019-01-03 | 2019-05-10 | 西北农林科技大学 | Direct immunization chromatographs detection method, test strips and the application for detecting Escherichia coli |
| WO2019133827A1 (en) * | 2017-12-29 | 2019-07-04 | Board Of Regents, The University Of Texas System | Antimicrobial nanobodies |
| CN110261608A (en) * | 2019-05-29 | 2019-09-20 | 江苏大学 | Food E. coli clones Visual retrieval and automated enumeration method based on magnetic fluorescence probe |
-
2019
- 2019-11-12 CN CN201911099944.8A patent/CN110988349B/en active Active
Patent Citations (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007037282A1 (en) * | 2005-09-27 | 2007-04-05 | Eisai R & D Management Co., Ltd. | Method of forming autoaggregate on microparticle and method of detecting target analyte |
| WO2009026141A1 (en) * | 2007-08-17 | 2009-02-26 | University Of Florida Research Foundation, Inc. | Supercrystalline colloidal particles and method of production |
| CN103116026A (en) * | 2013-01-29 | 2013-05-22 | 南昌大学 | Quick detection method for food-borne pathogens based on immunomagnetic separation of Fe3O4 nano materials |
| CN103383397A (en) * | 2013-06-24 | 2013-11-06 | 西北农林科技大学 | Alicyclobacillus immunomagnetic microspheres and application thereof |
| CN103439496A (en) * | 2013-08-13 | 2013-12-11 | 南昌大学 | Escherichia coli O157:H7 enrichment and rapid detection method |
| CN104749365A (en) * | 2013-12-31 | 2015-07-01 | 中国科学院上海微系统与信息技术研究所 | Difunctional composite nanosphere and method for rapidly detecting food-borne pathogenic bacteria |
| US20160172027A1 (en) * | 2014-12-16 | 2016-06-16 | Conversant Intellectual Property Management Inc. | Polymer Memory |
| CN106290882A (en) * | 2015-06-01 | 2017-01-04 | 上海凯创生物技术有限公司 | A kind of Candida albicans antigen near-infrared fluorescent detection kit and application thereof |
| CN105651991A (en) * | 2016-01-19 | 2016-06-08 | 南昌大学 | Method for rapidly detecting enterobacter sakazakii |
| CN105527428A (en) * | 2016-01-19 | 2016-04-27 | 南昌大学 | Method for fast detecting colon bacillus O157:H7 |
| CN106290320A (en) * | 2016-09-09 | 2017-01-04 | 南昌大学 | A kind of OTA chemical luminescence detection method based on unmarked aptamer sensor |
| CN107389919A (en) * | 2017-07-27 | 2017-11-24 | 广东省微生物研究所（广东省微生物分析检测中心） | A kind of label-free fluorescence aptamer sensor and its preparation method and application |
| CN108303532A (en) * | 2017-12-15 | 2018-07-20 | 浙江工商大学 | A kind of escherichia coli O157 of non-diagnostic purpose:The rapid detection method of H7 |
| CN108020674A (en) * | 2017-12-22 | 2018-05-11 | 天津现代职业技术学院 | It is a kind of to utilize the attached quick method for differentiating strain level pathogenic bacteria in food of double suction |
| WO2019133827A1 (en) * | 2017-12-29 | 2019-07-04 | Board Of Regents, The University Of Texas System | Antimicrobial nanobodies |
| CN108715889A (en) * | 2018-04-27 | 2018-10-30 | 曾小敏 | A kind of state enterorrhagia Bacillus coil 0157 living:The rapid detection method of H7 |
| CN109738638A (en) * | 2019-01-03 | 2019-05-10 | 西北农林科技大学 | Direct immunization chromatographs detection method, test strips and the application for detecting Escherichia coli |
| CN110261608A (en) * | 2019-05-29 | 2019-09-20 | 江苏大学 | Food E. coli clones Visual retrieval and automated enumeration method based on magnetic fluorescence probe |
Non-Patent Citations (4)
| Title |
|---|
| YING-DI ZHU 等: ""Fluorescent immunosensor based on CuS nanoparticles for sensitive detection of cancer biomarker"", 《ANALYST》 * |
| 王树坤等: "云南省首次检出O157大肠杆菌", 《疾病监测》 * |
| 蓝兰等: "沙门氏菌检测方法研究综述", 《广西畜牧兽医》 * |
| 邴欣等: "基于光热效应的沙门氏菌试纸条检测方法研究", 《食品研究与开发》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111948397A (en) * | 2020-06-09 | 2020-11-17 | 江苏海洋大学 | Double-antibody sandwich ELISA method for rapidly detecting Escherichia coli O157: H7 |
| CN111948397B (en) * | 2020-06-09 | 2023-03-10 | 江苏海洋大学 | Double-antibody sandwich ELISA method for rapidly detecting Escherichia coli O157H 7 |
| CN114113585A (en) * | 2021-11-04 | 2022-03-01 | 西北农林科技大学 | Double-signal probe, test strip for detecting escherichia coli and application |
| CN114113585B (en) * | 2021-11-04 | 2023-10-31 | 西北农林科技大学 | Double-signal probe, test strip for detecting escherichia coli and application |
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