CN110988349B - Capture probe, two-channel detection method of Escherichia coli O157: H7 and application thereof - Google Patents
Capture probe, two-channel detection method of Escherichia coli O157: H7 and application thereof Download PDFInfo
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- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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Abstract
The invention discloses a capture probe, a two-channel detection method of Escherichia coli O157: H7 and application thereof, wherein the capture probe is Fe 3 O 4 (ii)/CuS, said Fe 3 O 4 the/CuS includes first preparing Fe 3 O 4 Micro-spheres and then mixing Fe 3 O 4 Adding microsphere into CTAB solution, adopting chemical deposition method, adding CuSO 4 With Na 2 S solution is stirred and washed to prepare Fe 3 O 4 and/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 Fe 3 O 4 the/CuS nano material has a photo-thermal characteristic, and on the basis of visual 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 capture probe, a double-channel detection method of Escherichia coli O157: H7 and application thereof.
Background
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 O157H 7 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 O157H 7 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) Although the traditional materials have advantages, the traditional materials are limited to the limitation of complex crosslinking between the materials and the antibodies; (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 form 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 Fe 3 O 4 (ii) CuS, said Fe 3 O 4 the/CuS includes first preparing Fe 3 O 4 Micro-spheres and then mixing Fe 3 O 4 Adding microsphere into CTAB solution, adopting chemical deposition method, adding CuSO 4 With Na 2 S solution is stirred and washed to prepare Fe 3 O 4 /CuS。
Further, said Fe 3 O 4 The 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 added 3 O 4 Adding the microspheres into CTAB solution, stirring, and suspending in Na 2 Adding CuSO into the S aqueous solution dropwise 4 Continuously stirring the aqueous solution, washing and drying to obtain the water-soluble organic fertilizer.
A dual-channel detection method for 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 the 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 808nm.
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 O157H 7 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 O157H 7 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 potassium dihydrogen phosphate adding 0.02g of water and fixing the volume to 100mL to obtain the product.
Specifically, the detection limit of the test strip on Escherichia coli O157H 7 is 10 2 CFU/mL。
The capture probe is used for detecting Escherichia coli O157: H7 in beef, chicken, milk and honey.
The two-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 Fe 3 O 4 the/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 7 2 CFU/mL, the value is lower than that reported in many other documents.
(5) The specificity is high. The monoclonal antibody prepared by using bacterial flagellin immunization can only recognize Escherichia coli O157: H7 with high specificity, and has no specificity to other phyla bacteria.
(6) Good practical application. The method 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 test strip for detecting Escherichia coli O157H 7 according to the present invention;
FIG. 2 is a schematic flow chart of the immunochromatography for rapidly detecting Escherichia coli O157H 7 according to the present invention;
FIG. 3 shows the optimized result of the immunochromatographic test strip prepared in 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 the verification of the novel probe prepared by 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 the recently discovered photothermal effect is used for enhancing the sensitivity of an immunochromatographic test strip and photothermal materials, and the results of the photothermal nanomaterials enable the current research to propose photothermal immunoassay and construct a few 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 O157H 7. The invention relates to a double-channel detection method, which comprises the following steps: in one aspect, fe 3 O 4 The novel unlabeled immunochromatographic test strip detection strategy only uses one antibody, the bacteria serves as an intermediate for connecting materials and the antibody together, the cost is saved, and high-sensitivity detection is realized. On the other hand, based on the photothermal effect of the material, 808nm near red is usedA simple sensor device is manufactured by the external light laser pen, the infrared camera and the smart phone, near infrared light irradiates materials accumulated on a T line, the temperature difference (delta T) before and after irradiation of each test strip T line is recorded, and the accuracy and the sensitivity of the biosensor are further improved. Coli O157H 7 in the sample was first Fe 3 O 4 the/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, fe 3 O 4 The 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 invention 3 O 4 The term "CuS" refers to the diamond-shaped cluster-shaped CuS material and the spherical magnetic nano-material Fe prepared by the preparation method 3 O 4 And compounding.
The working principle of the invention is as follows: based on Fe 3 O 4 the/CuS nano material can directly adsorb bacteria, and Fe is added during detection 3 O 4 the/CuS was added directly to a bacterial suspension of E.coli O157: H7, which was immediately adsorbed on the bacterial surface. And then, adding the novel probe compound on a sample pad, and drawing the sample pad by capillary force to absorb the probe compound, wherein the monoclonal antibody can be specifically combined with the Escherichia coli O157: H7 antigen, and the Escherichia coli O157: H7 monoclonal antibody fixed on the T line captures the compound. 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 perform the 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 lines, the change of temperature is measured through a photothermal converter, more nanometer materials combined with bacteria are accumulated on the T lines along with the increase of the concentration of the compound, the change of temperature is more obvious, and therefore photothermal detection of the bacteria is achieved.
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 detection limit of sensitivity 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 O157H 7 monoclonal antibody may be a monoclonal antibody prepared by a conventional method or commercially available, and is preferably prepared according to the method described in Zhang Daohong et al, publication of Analytical chip Acta, volume 635, page 63-69, entitled "Production of an exogenous 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 antiserum for resisting Escherichia coli to generate, performing semisolid cell fusion on splenocytes of the mouse and 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 matched 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 synthesis 3 ·6H 2 O (1.35 g) and citric acidTrisodium (0.45 g) was mixed in ethylene glycol (40 mL) and stirring continued for 60 minutes, naAc (2.4 g) 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 Fe 3 O 4 And (4) microspheres.
(2) Adopting a chemical deposition method to obtain Fe obtained in the step one 3 O 4 (0.2 g) microspheres were added to CTAB solution (0.5 g/L25 mL cetyltrimethylammonium bromide), stirred for 0.5 h, the solution was washed three times, stirred at room temperature for 3 h, suspended in Na 2 Aqueous S solution (0.04M, 25mL) and then CuSO was added dropwise 4 The 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 Fe 3 O 4 The 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 Escherichia coli O157: H7 flagellin solution and an equal amount of Freund 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 is carried out, 0.8mg/mL escherichia coli O157: H7 flagellin is mixed and emulsified with an equal amount of Freund incomplete adjuvant, and the emulsified antigen is injected into the abdominal cavity of a mouse. 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 in dose, and each rat is 100 mug of Escherichia coli O157H 7; 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 the 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 1450 g/mol) as a fusion agent to carry out cell fusion according to a conventional method. The specific method comprises the following steps:
(1) Taking 1-2 x 10 7 Mixing 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 CO 2 Taking out 50 percent PEG which is incubated to 37 ℃ from an incubator, sucking 0.8mL by using a 1mL suction tube, holding a 50mL centrifuge tube filled 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 the PEG, continuously adding the PEG 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 the PEG to the fused cells, gently stirring while adding the PEG to disperse cell masses, 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 down a cover and reversing for several times to uniformly 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) The resuspended cells were added to 80mL of cells incubated at 37 deg.CMixing the cells in the semi-solid culture medium by gentle shaking, sucking up the semi-solid culture medium with the fused cells by a 20mL syringe, uniformly distributing the semi-solid culture medium into 8-9 six-hole cell culture plates with 1.5-2 mL/hole, and placing the cells in a CO culture plate with 37 DEG C 2 Culturing 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-well culture plate, wherein each well is provided with one clone. The culture was continued with HT liquid medium. When cloning to reach 1/2-2/3 of the full bottom of the hole, the culture solution turns yellow, and then the hybridoma cell can be screened.
Step three: screening of cell lines
Screening out positive holes for resisting Escherichia coli O157H 7 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 O157H 7, 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 carried out by the following specific procedures: the ascites fluid was filtered with double-layer filter paper, centrifuged at 12000r/min at 4 ℃ for 15min, and the supernatant was aspirated. The resulting ascites supernatant was mixed with 2 times the volume of acetate buffer, 33. Mu.L/mL of ascites in n-octanoic acid was slowly added with stirring, mixed at room temperature for 30min, and allowed to stand at 4 ℃ for 2 hours. Centrifuging at 12000r/min for 30min at 4 ℃, and discarding the precipitate. After the supernatant was filtered through a 0.22 μm filter, 0.1mol/L of a phosphate buffer solution having a pH of 7.4 was added to the filtrate in an amount of 1/10 by volume, and the pH of the mixture was adjusted to 7.4 with 2mol/L of a 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 by 0.02mol/L phosphate buffer solution with the volume of 1/10 of the original ascites volume, and is put into a dialysis bag, firstly dialyzed for 6-8h by 0.01mol/L phosphate buffer solution, and then 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 the purified Escherichia coli O157: H7 monoclonal antibody, and placing the antibody in the 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 to be 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 80g of sodium hydroxide, and water is added to the sodium hydroxide solution to be constant volume of 1000 mL.
Escherichia coli O157 used in the following examples 3 to 5H 7 general monoclonal antibody was prepared as described in example 2.
Example 3: this example presents a condition-optimized experiment for a 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 comprises: 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 the length of 15mm and the width of 3mm, soaking the pieces in a sealing solution, drying the pieces at 37 ℃ for 10-16 hours to obtain a sample pad, and then placing the sample pad in a dryer for storage 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 in 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; the cells were washed 3 times with 0.01mol/L phosphate buffer solution (pH7.4), and resuspended in 10mL0.01 mol/L phosphate buffer solution; adding 0.5% formalin solution, 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 10 8 CFU/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)Fe 3 O 4 Optimization of CuS solution concentration
Fe 3 O 4 The concentration of/CuS has a significant influence on the coloring effect of the bacteria. At a fixed bacterial concentration of 10 8 Different Fe were studied under CFU/mL 3 O 4 The concentration of/CuS was 0.5,1.0,1.5 and 2.0mg/mL, respectively.
(8)Fe 3 O 4 Optimization of/CuS solution volume
Fe 3 O 4 The 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 10 to 80. Mu.L and 100. Mu.L of 10 6 CFU/mL bacterial solution mixed incubation and test.
(9) Time of immune response
Another important parameter that affects the intensity of the detection line is the immunoreaction time. The result was measured every 5 minutes for 5-15 minutes after the addition of the stained bacteria to the sample pad.
(10)Fe 3 O 4 Optimization of incubation time of CuS solution with bacterial solution
The time of incubation may also affect the signal intensity on the detection line. Mixing Fe 3 O 4 the/CuS and bacteria mixture was incubated for different times in the range of 10, 60, 120, 180, 240, 360, 600, 900s.
The results are shown in FIG. 3A, with Fe 3 O 4 And the color intensity of the detection line is in an increasing trend due to the increase of the concentration of/CuS. When Fe 3 O 4 The 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 Fe 3 O 4 Volume increase of/CuS, volume increased to 40. Mu.L, reaching maximum. Thus, fe 3 O 4 The 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, fe 3 O 4 the/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 Fe 3 O 4 CuS is dissolved in water to prepare a solution with the concentration of 1.0mg/mL, and then the bacterial liquid is diluted into 80-10 parts by 0.01M phosphate buffer solution 8 CFU/mL concentration, 100. Mu.L of each solution was taken as the detection solution, and 40. Mu.L of Fe 3 O 4 CuS mixed incubation for 4 minutes, then dropwise adding the sample pad of the test strip, and taking 100 mu L0.01M phosphate buffer as negativeThe control, operating as above, was added drop wise to the sample pad of another test strip 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, which indicates that the concentration of Escherichia coli O157H 7 in the sample to be detected is higher than or equal to 10 3 CFU/mL. (2) negative: when the detection line of the detection test strip does not display color, the result is negative, which indicates that the ratio of Escherichia coli O157H 7 in the sample to be detected is less than 10 3 CFU/mL。
As shown in FIG. 4A, as the concentration of E.coli O157H 7 decreased, the test strip T line became lighter in brown color and the macroscopic concentration was 10 3 CFU/mL, therefore, the lowest concentration of Escherichia coli O157H 7 which can be detected by naked eyes is 10 3 CFU/mL。
As shown in FIG. 4B, as the concentration of Escherichia coli O157H 7 is reduced, the detected photothermal temperature is lower, and the concentration detected by the photothermal test strip is 10 2 CFU/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 paper strip preparation and the procedure of the bacterial culture process were the same as those in (1) to (6) of example 2.
The specific detection process comprises the following steps: mixing Fe 3 O 4 the/CuS is dissolved in water to prepare a solution with the concentration of 1.0mg/mL, and bacterial liquids of Escherichia coli O157H 7, salmonella typhimurium, salmonella hadamara, salmonella london, staphylococcus aureus, campylobacter jejuni, listeria monocytogenes and Salmonella enteritidis are respectively diluted into 10 by 0.01M phosphate buffer solution 8 CFU/mL concentration, 100. Mu.L solution was taken as the detection solution for each concentration, and 40. Mu.L Fe 3 O 4 the/CuS mixture was incubated for 4 minutes, and then the sample pad of the test strip was added dropwise, and at the same time, 100. Mu. L0.01M phosphate buffer was used as a negative control, and the same procedure as above was followed, and the sample pad of the other 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 the result as negative.
Referring to FIG. 5, numerals 1-9 each represent 10 8 CFU/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. Except that the test strip T line for detecting the Escherichia coli O157H 7 has bright brown color which can be seen by naked eyes, the test strip T lines for detecting other bacteria have no color, which indicates that the invention can highly specifically recognize the Escherichia coli O157H 7 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 Fe 3 O 4 Dissolving CuS in water to obtain solution with concentration of 1.0mg/mL, adding bacterial solution with known concentration into drinking water to form 80-10 8 CFU/mL concentration, 100. Mu.L of each solution was taken as the detection solution, and 40. Mu.L of Fe 3 O 4 CuS is mixed and incubated for 4 minutes, then the sample pad of the test strip is added dropwise, meanwhile, 100 mu L of drinking water is taken as a negative control solution, the operation is the same as the above, the sample pad of the other test strip is added dropwise, the result is read after 10 minutes, and then the temperature difference is obtained by irradiating T rays with near infrared light of 808nm.
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 the result as negative.
As shown in figure 6, as the concentration of bacteria is reduced, the brown color on the test strip becomes lighter and lighter, and the macroscopic detection limit concentrations of actual samples of beef, chicken, milk and honey can reach 10 respectively 4 ，10 3 ，10 4 ，10 3 CFU/mL, photothermal detection limit concentration of 10 3 ，10 2 ，10 3 ，10 2 CFU/mL reflects the good practical application value.
Example 7, fe 3 O 4 Characterization of the/CuS Material
To demonstrate the use of Fe in the present invention 3 O 4 The inventors also performed the following experiments:
(1) Scanning electron microscope: FIG. 7A is Fe 3 O 4 A scanning electron microscope picture is spherical, and the particle size is 220-280 nm; FIG. 7B is Fe 3 O 4 Electron microscope image of/CuS composite nano material, rhombic cluster material CuS (240-420 nm) and spherical magnetic nano material Fe 3 O 4 The composite material is formed; FIG. 7C is Fe 3 O 4 The 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 Fe 3 O 4 the/CuS composite nanomaterial can capture and kill bacteria, and fig. 7D insets a and b show that the a diagram is the bacterial solution with OD600=1 in the absence of any external magnetic field, and the b diagram is the bacterial solution with OD600=1 and Fe 3 O 4 Mixed solution of/CuS, beside a magnet, in the absence of any external magnetic field, a solution of bacteria with OD600=1.0 was mixed with Fe 3 O 4 After the/CuS mixture, the mixture was almost transparent within 30 minutes after the magnet was applied.
(2) Bacteriostatic map: 7E is Fe 3 O 4 The antibacterial tests of/CuS and three bacteria are implemented by using salmonella enteritidis, escherichia coli O157: H7 and Listeria in test detection, and the number of bacteria in experimental groups (a, b and c) is observed to be 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 Fe 3 O 4 In contrast, nanocomposite Fe 3 O 4 The characteristic peak of/CuS (dotted line) is deep red shifted 570nm.
(4) Infrared characterization: as can be seen from FIG. 8A, fe 3 O 4 the/CuS nano composite material not only has the characteristic Cu-S peak (1110 cm) of CuS -1 ) And Fe 3 O 4 Fe-O peak of (594 cm) -1 )，1637cm -1 And 1401cm -1 Spectral band of (b) can be attributed toStretching vibration of COOH (1).
(5) X-ray photoelectron spectroscopy: from fig. 8B, it can be seen that at 934.16, 530.41, 168.79, 284.94 and 711.34eV, there are five different peaks on the curves 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, fe 3 O 4 the/CuS nanocomposite has both the characteristic CuS peaks at 29.08 ° (102), 31.54 ° (103), 34.57 ° (006), 49.56 ° (110) and 57.98 ° (116), and Fe 3 O 4 32.40 ° (220), 36.57 ° (331), 43.20 ° (400), 54.87 ° (422), 56.30 ° (511) and 62.17 ° (440), proving the success of composite synthesis.
(7) And (3) photothermal characterization: from FIG. 8D, following Fe 3 O 4 The 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 (6)
1. 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 a capture probe, incubating to obtain a liquid to be detected, dripping the liquid to be detected onto a test strip for detecting the Escherichia coli O157H 7, and irradiating the test strip by adopting near infrared light to realize dual-channel detection;
the capture probe is Fe 3 O 4 (ii)/CuS, said Fe 3 O 4 the/CuS includes first preparing Fe 3 O 4 Micro-spheres and then mixing Fe 3 O 4 Adding microsphere into CTAB solution, adding CuSO by chemical deposition method 4 With Na 2 S solution is stirred and washed to prepare Fe 3 O 4 /CuS；
Said Fe 3 O 4 The addition amount of the microspheres is 0.1-0.3 g, and the particle size is 220-280 nm;
the particle size of the CuS is 240-420 nm.
2. The dual-channel detection method of Escherichia coli O157: H7 as claimed in claim 1, 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 808nm.
3. The dual-channel detection method of Escherichia coli O157H 7 as claimed in claim 1, 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 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 O157H 7 monoclonal antibody, the binding pad and the sample pad are respectively sealed by sealing liquid, and the test strip has no quality control line.
4. The dual-channel detection method of Escherichia coli O157H 7 as claimed in claim 3, wherein the detection line coated with Escherichia coli O157H 7 monoclonal antibody is prepared by 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 muL/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 and sodium chloride injection solution is obtained.
5. The dual-channel detection method for Escherichia coli O157H 7 as claimed in claim 3, wherein the detection limit of the test strip for Escherichia coli O157H 7 is 10 2 CFU/mL。
6. The dual-channel detection method of Escherichia coli O157: H7 as claimed in any one of claims 1-5 is used for detecting Escherichia coli O157: H7 in beef, chicken, milk and honey.
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