Immunochromatography test strip for rapidly detecting escherichia coli O157: H7 through AIE double labeling and high sensitivity and application of immunochromatography test strip

Abstract

The invention discloses an AIE double-labeled high-sensitivity immunochromatographic test strip for rapidly detecting escherichia coli O157: H7 and application thereof, and belongs to the technical field of escherichia coli detection. The immunochromatography test strip consists of a sample pad, a combination pad, a nitrocellulose membrane and an absorption pad which are sequentially connected and fixed on a bottom plate; an escherichia coli O157: H7 detection antibody marked by TCBPE is coated on the combination pad; the nitrocellulose membrane is sequentially coated with a detection line T and a quality control line C which are separated from each other; the detection line T is coated with an escherichia coli O157: H7 capture antibody; and the quality control line C is coated with a goat anti-mouse antibody. The immunochromatography test strip has the advantages of high sensitivity and accuracy, strong stability and convenience in operation. The invention provides a new method for detecting escherichia coli O157: H7.

Description

Technical Field

[0001] The present invention relates to the technical field of Escherichia coli detection, and particularly to an immunochromatographic test strip for highly sensitive and rapid detection of Escherichia coli O157:H7 with AIE dual labeling and its application. Background Art

[0002] Escherichia coli O157:H7 is an enterohaemorrhagic Escherichia coli and one of the main foodborne pathogenic bacteria. The intestinal infectious disease caused by Escherichia coli O157:H7 has become a global public health concern. The infection of Escherichia coli O157:H7 can cause severe gastrointestinal complications such as hemorrhagic colitis (HC), appendicitis and colon perforation, and can seriously cause systemic complications such as thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS). Among them, 3-5% of patients die, and about 12% of patients have serious sequelae. Moreover, the infectious dose in humans is extremely low, and ingestion of 10-100 live bacteria can cause disease. In recent years, the detection rate of Escherichia coli O157:H7 in food samples in China, especially in animal meat products, is relatively high. This bacterium has been detected in various foods in Fujian, Beijing, Guangdong and other provinces and cities. Escherichia coli O157:H7 has strong adaptability to the external environment, has certain resistance to temperature, pH and dryness, and can survive in food, sand, water, fertilizer and artificial microenvironment for up to several months.

[0003] At present, the detection technologies of Escherichia coli O157:H7 mainly include the following categories: Traditional culture method (gold standard): It needs to go through steps such as enrichment culture, selective isolation, and biochemical identification. It takes a long time (usually 24-72 hours), the operation is cumbersome, and it cannot meet the requirements of rapid detection. Immunological methods: ① Colloidal gold test strip: Based on antigen-antibody reaction, the detection limit is about 10 5-6 CFU / mL, the sensitivity is relatively low, and the colloidal gold label is easy to aggregate and has poor stability, resulting in large differences between batches. ② Latex agglutination method: Agglutination reaction is carried out by coating antibodies on latex particles, but the specificity is easily interfered by food matrix, and the requirement for colony concentration is high (≥10 6 CFU / mL). ③ Enzyme-linked immunosorbent assay (ELISA): It requires multiple washing and color development operations, the detection time is still relatively long (4-6 hours), and it depends on the activity stability of enzyme labels. Molecular biology methods (such as PCR): The sensitivity can reach 10 2 CFU / mL, but it requires complex pretreatment (DNA extraction) and precision instruments, and is easily affected by inhibitors in food, and cannot achieve on-site rapid detection.

[0004] Moreover, the test strips in the prior art have the following defects: (1) Insufficient sensitivity: The detection sensitivity of traditional colloidal gold test strips is poor (105 -10 6 CFU / mL), it is difficult to meet the detection requirements for low - concentration contamination (usually ≤ 10 4 CFU / mL) in food. (2) Poor stability of labeling materials: Colloidal gold is prone to aggregation, leading to signal attenuation; quantum dots are easily affected by photo - oxidation; latex particles are prone to non - specific adsorption, all of which affect the detection reproducibility. (3) Dependence on pre - enrichment steps: Most methods require more than 12 hours of pre - enrichment culture to enrich the target bacteria, resulting in an extended detection cycle. (4) Risk of cross - reaction: For example, the latex agglutination method may have a cross - reaction with Escherichia coli of non - O157:H7 serotype.

[0005] Therefore, improving the detection sensitivity, enhancing the stability of labeling materials, shortening the detection time, and improving the specificity have become urgent problems to be solved in the current detection of Escherichia coli O157:H7. Summary of the Invention

[0006] The object of the present invention is to provide an immunochromatographic test strip for highly sensitive and rapid detection of Escherichia coli O157:H7 with AIE dual - labeling and its application, so as to solve the problems existing in the above - mentioned prior art. The immunochromatographic test strip of the present invention has the advantages of high sensitivity and accuracy, strong stability, and operational convenience, providing a new method for the detection of Escherichia coli O157:H7.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides an immunochromatographic test strip for highly sensitive and rapid detection of Escherichia coli O157:H7 with AIE dual - labeling, which is composed of a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad that are sequentially connected and fixed on a bottom plate;

[0009] The conjugate pad is coated with an Escherichia coli O157:H7 detection antibody labeled with TCBPE;

[0010] The nitrocellulose membrane is sequentially coated with a separated test line T and a control line C;

[0011] The test line T is coated with an Escherichia coli O157:H7 capture antibody;

[0012] The control line C is coated with a goat anti - mouse antibody.

[0013] Optionally, the Escherichia coli O157:H7 detection antibody labeled with TCBPE includes a monoclonal antibody against Escherichia coli O157:H7.

[0014] Optionally, the preparation method of the Escherichia coli O157:H7 detection antibody labeled with TCBPE includes the following steps:

[0015] Add the TCBPE solution to the boric acid buffer solution, then add the EDC solution and the NHS solution to activate the carboxyl group of TCBPE, centrifuge and resuspend the precipitate to obtain the carboxyl-activated TCBPE solution;

[0016] Adjust the concentration of the Escherichia coli O157:H7 detection antibody to 1 mg / mL, add 2 mL of the carboxyl-activated TCBPE solution for labeling, then add the blocking solution for blocking, centrifuge and take the precipitate, and ultrasonically resuspend and wash to obtain the product;

[0017] The volume ratio of the boric acid buffer solution, the TCBPE solution, the EDC solution and the NHS solution is 20:1:2:1.5.

[0018] Optionally, the test line T and the control line C are parallel to each other and have an equal width interval, and the interval is 0.5 cm.

[0019] Optionally, the Escherichia coli O157:H7 capture antibody includes the Escherichia coli O157:H7 monoclonal antibody.

[0020] Optionally, the bottom plate includes a PVC adhesive bottom plate.

[0021] The present invention also provides the application of the immunochromatographic test strip in detecting Escherichia coli O157:H7.

[0022] The present invention also provides a method for detecting Escherichia coli O157:H7, including the step of detecting by using the immunochromatographic test strip.

[0023] Optionally, it further includes the step of pre-enriching the sample.

[0024] Optionally, the pre-enrichment of the sample includes: adding the sample to a nutrient broth liquid medium, then adding TCBPE, and culturing for 4-5 h.

[0025] The present invention discloses the following technical effects:

[0026] The present invention establishes an immunochromatographic test strip based on the AIE characteristic material 1,1,2,2-Tetra(4-carboxylbiphenyl)ethylene (TCBPE) that labels both the antigen of Escherichia coli O157:H7 and the monoclonal antibody of Escherichia coli O157:H7. TCBPE has the advantages of high photostability, large Stokes shift, low background, high fluorescence efficiency, easy modification of molecular functional groups, and stable properties. In the actual detection water environment, TCBPE will rapidly aggregate and emit strong fluorescence. Through the double amplification of the fluorescence signal, the high-sensitivity detection of Escherichia coli O157:H7 by the immunochromatographic test strip is realized. The test strip mainly consists of a sample pad, a conjugate pad, a nitrocellulose membrane (NC membrane), an absorbent pad, and a PVC bottom plate. Specifically: (1) The capture antibody of Escherichia coli O157:H7 and the goat anti-mouse antibody are respectively sprayed in parallel on the NC membrane as the test line (T line) and the control line (C line). The detection antibody of Escherichia coli O157:H7 labeled with TCBPE is sprayed on the conjugate pad. Based on the double-antibody sandwich mode formed by the two monoclonal antibodies of the capture antibody and the detection antibody, the specificity of the test strip detection is improved, and the cross-reaction with 23 other bacteria is eliminated; (2) TCBPE is added during the sample enrichment process. While the target bacteria are enriched, they are fluorescently stained and labeled by TCBPE. Through the double labeling of the antigen and the antibody by AIE, the fluorescence signal is amplified, and the high-sensitivity detection of Escherichia coli O157:H7 by the test strip is realized. The present invention provides a new method for the detection of Escherichia coli O157:H7.

[0027] The test strip of the present invention has the following advantages: High sensitivity and accuracy: The detection limit is 10 4 CFU / mL, which can detect trace contamination in food, comparable to the PCR method, but without the need for complex instruments. Strong stability: The TCBPE material is resistant to high temperature and repeated freeze-thaw cycles, suitable for field and cold chain environments. Operational convenience: No professional training is required, and on-site rapid screening is supported, suitable for large-scale food safety supervision. Policy compliance: It meets the requirements of "biotechnology innovation empowering food safety" and provides technical support for the quality control of the food industry chain. Brief Description of the Drawings

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0029] Figure 1Schematic diagram of the AIE dual-labeled highly sensitive and rapid detection immunochromatographic test strip for Escherichia coli O157:H7 in the embodiments of the present invention; wherein 1: sample pad, 2: conjugate pad, 3: TCBPE-labeled detection antibody for Escherichia coli O157:H7, 4: capture antibody for Escherichia coli O157:H7, 5: nitrocellulose membrane (NC membrane), 6: goat anti-mouse antibody, 7: absorbent pad, 8: test line (T line), 9: control line (C line), 10: PVC adhesive bottom plate;

[0030] Figure 2 Schematic diagram of the detection process and result determination in the embodiments of the present invention; a: detection process; b: observing results; c: result determination;

[0031] Figure 3 Observation results of Escherichia coli O157:H7 stained with TCBPE under a fluorescence microscope in the embodiments of the present invention;

[0032] Figure 4 Results of the detection sensitivity of the TCBPE dual-labeled immunochromatographic test strip in the embodiments of the present invention, T is the test line, C is the control line, 9-2 successively represent the bacterial concentration of 10 9 -10 2 CFU / mL, N is the negative control;

[0033] Figure 5 Results of the specificity determination of the TCBPE dual-labeled immunochromatographic test strip in the embodiments of the present invention, T is the test line, C is the control line, 1-26 respectively correspond to the strains of each serial number in Table 1;

[0034] Figure 6 Results of the simulated bacteria-carrying detection of the TCBPE dual-labeled immunochromatographic test strip for Chinese cabbage samples in the embodiments of the present invention, 1-12 are the enrichment time (h), N is the negative control;

[0035] Figure 7 Results of the simulated bacteria-carrying detection of the TCBPE dual-labeled immunochromatographic test strip for milk samples in the embodiments of the present invention, 1-12 are the enrichment time (h), N is the negative control;

[0036] Figure 8 Results of the simulated bacteria-carrying detection of the TCBPE dual-labeled immunochromatographic test strip for beef samples in the embodiments of the present invention, 1-12 are the enrichment time (h), N is the negative control. Detailed implementation manners

[0037] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be construed as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0038] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0039] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0040] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the present invention specification are obvious to those skilled in the art. The present invention specification and examples are merely exemplary.

[0041] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0042] Example 1

[0043] 1. Main reagents

[0044] Tween-20 and polyvinylpyrrolidone K-30 were purchased from Sinopharm Chemical Reagent Co., Ltd.; TCBPE was purchased from Zhengzhou Alpha Chemical Co., Ltd.; 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; sample pads, conjugate pads, absorbent pads, and PVC bottom plates were purchased from Shanghai Goldbio Technology Co., Ltd.; BSA was purchased from Shanghai Jieyi Biotechnology Co., Ltd.; nitrocellulose membranes were purchased from Sartorius.

[0045] 2. Main instruments

[0046] The microplate reader SpectraMax M2 was purchased from Molecular Devices; the magnetic heating stirrer was purchased from Suzhou Sains Instruments Co., Ltd.; the spotting instrument AD6010 was purchased from BIO-DOT; the automatic chopping machine ZQ2000 and the plate pasting machine TB 300 were purchased from Shanghai Gold Label Biotechnology Co., Ltd.; the combined shaking incubator ZQPZ 228R was purchased from Labotery Instrument Equipment Co., Ltd.; the single-person purification workbench SW-SJ-2D was purchased from Suzhou Purification Equipment Co., Ltd.

[0047] 3. Experimental strains

[0048] The experimental strains used in this example are shown in Table 1. Each strain was purchased from the corresponding depository institution.

[0049] Table 1 Experimental strains

[0050]

[0051] 4. Construction of the AIE dual-labeled test strip

[0052] 4.1 Test strip construction

[0053] As Figure 1 shown, the highly sensitive and rapid detection immunochromatographic test strip for Escherichia coli O157:H7 has the following characteristics: It consists of a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad that are sequentially connected and fixed on the bottom plate. The conjugate pad is coated with a TCBPE-labeled detection antibody for Escherichia coli O157:H7 (in this example, a monoclonal antibody against Escherichia coli O157:H7 is used); on the nitrocellulose membrane, a separated test line T and a control line C are sequentially coated; the test line T is coated with a capture antibody for Escherichia coli O157:H7 (in this example, a monoclonal antibody against Escherichia coli O157:H7 is used); the control line C is coated with a goat anti-mouse antibody.

[0054] The preparation method is as follows:

[0055] Preparation of TCBPE-labeled Escherichia coli O157:H7 detection antibody: 1. Carboxyl activation of TCBPE: Add 30 mL of boric acid buffer (0.2 mol / L, pH 8.61) to a conical flask. While stirring, add 1.5 mL of TCBPE solution (0.006 mol / L, solvent DMSO), then add 3 mL of freshly prepared EDC solution (10 mg / mL) and 2.25 mL of freshly prepared NHS solution (10 mg / mL). Stir in the dark for 2 h. Transfer the solution to a centrifuge tube and centrifuge at 12000 rpm for 20 min. Discard the supernatant. Resuspend and dissolve the precipitate with 1 mL of DMSO and store it in the dark at room temperature. 2. Cross-linking and labeling of carboxyl-activated TCBPE with Escherichia coli O157:H7 detection antibody: Pipette 1 mL of Escherichia coli O157:H7 detection antibody solution (5.2 mg / mL) into a glass bottle. Dilute the antibody to 1 mg / mL with phosphate buffer (PB, 0.01 mol / L, pH 8.2). Place the glass bottle on a magnetic stirrer and stir at a speed of 150 rpm at 4 °C. Dropwise add 2 mL of TCBPE carboxyl-activated solution and stir in the dark for 12 h for labeling. Finally, add 2 mL of BSA blocking solution to a final concentration of 3% and continue to stir in the dark at 4 °C overnight. After blocking, transfer the solution to a centrifuge tube and centrifuge at 14000 rpm at 4 °C for 20 min. Resuspend and wash the precipitate with 2 mL of PB by ultrasonic treatment twice and store it in the dark at 4 °C for standby.

[0056] Preparation of test strip: Spray the TCBPE-labeled Escherichia coli O157:H7 detection antibody prepared above onto the conjugate pad using a spotting instrument. Spray 1 mg / mL of Escherichia coli O157:H7 capture antibody and goat anti-mouse antibody onto the NC membrane (2.5 cm × 30 cm) in parallel at 1 μL / cm using a spotting instrument as the test line and the control line, respectively. The distance between the test line and the control line is 0.5 cm. After spraying, place the NC membrane in the dark and dry it at 4 °C for storage. Place the treated NC membrane, conjugate pad (0.5 cm × 30 cm), sample pad (2 cm × 30 cm), and absorbent pad (2 cm × 30 cm) on the test strip laminator and paste them on the PVC adhesive bottom plate (6 cm × 30 cm) in an overlapping manner of 1 - 2 mm as shown Figure 1 and cut into test strips 3 mm wide using a test strip cutter and store them in the dark and dry at 4 °C.

[0057] 4.2 Principle and result determination of the detection process of AIE dual-labeled immunochromatographic test strip

[0058] The detection process of the AIE dual-labeled immunochromatographic test strip is as follows: Take 25 g or 25 mL of the food sample to be tested according to the national standard method and place it in 225 mL of sterile nutrient broth liquid medium. Add TCBPE and incubate at 37 °C for 5 h. Then, pipette 100 μL of the culture solution and drop it onto the sample pad of the test strip. As Figure 2As shown, observe the color development of the test line and the control line on the test strip under ultraviolet light.

[0059] The detection process and result judgment are as Figure 2 shown. When the sample is dropped on the sample pad of the test strip, the solution chromatographs towards the NC membrane by capillary action. If the sample solution does not contain stained Escherichia coli O157:H7, the test line will not show color, while the detection antibody labeled with TCBPE will still be captured by the goat anti-mouse antibody at the control line, making the control line show a green band, which is a negative result; if the sample solution contains stained Escherichia coli O157:H7, it will be captured by the capture antibody on the test line, making the test line of the test strip show color, and at the same time the control line also shows color, which is a positive result.

[0060] 5. Observation of TCBPE-stained Escherichia coli O157:H7 under a fluorescence microscope

[0061] Absorb the fresh Escherichia coli O157:H7 bacterial solution cultured for 5 h, centrifuge at 5000 rpm for 3 min, discard the culture solution, resuspend with an equal volume of ultrapure water, and drop it onto the glass slide; after the bacteria are dried and fixed, drop the TCBPE solution, let it stand for 1 min, then rinse the dye solution with ultrapure water, dry it and observe it under a fluorescence microscope. As Figure 3 can be seen, under the fluorescence microscope, fluorescence of Escherichia coli O157:H7 can be observed, indicating that TCBPE can effectively stain Escherichia coli O157:H7, making it show a bright green fluorescence signal.

[0062] Example 2 Determination of the detection sensitivity of the AIE dual-labeled immunochromatographic test strip

[0063] Culture Escherichia coli O157:H7 with fluorescence coloring to more than 10 9 CFU / mL, dilute it 10-fold with ultrapure water in gradient, take 100 μL of each gradient bacterial solution and drop it onto the sample pad of the test strip respectively, and use the stained culture medium as a negative control. The detection results are as Figure 4 shown. When the concentration of Escherichia coli O157:H7 is 10 9 -10 4 CFU / mL, there is a green fluorescent band at the test line and the intensity gradually weakens; when the concentration is 10 3 CFU / mL and 10 2 CFU / mL, no fluorescent band appears at the test line, which is consistent with the negative control, and the control line of all test strips shows color. The results show that the detection sensitivity of the TCBPE dual-labeled immunochromatographic test strip for detecting Escherichia coli O157:H7 is 10 4 CFU / mL.

[0064] Example 3 Cross-reaction determination of the AIE dual-labeled immunochromatographic test strip

[0065] The cross-reaction of 26 strains of foodborne pathogenic bacteria was determined using the dual-label test strip of the present invention. All the strains in Table 1 were cultured with TCBPE coloring until the concentration reached 10 8 CFU / mL or above, and the test strip of the present invention was used to detect each bacterial liquid to evaluate the specificity of the detection of the present invention.

[0066] As Figure 5 shown, when detecting Escherichia coli O157:H7 (ATCC 43889, ATCC 43895, and NCTC 12900) of strains 3, 4, and 5, green fluorescent bands appeared at the test line and the quality control line of the test strip respectively, and the result was judged as positive; while the results of detecting other foodborne pathogenic bacteria showed that green fluorescent bands only appeared at the quality control line and the test line did not show color, and the result was judged as negative. The results indicate that the dual-label immunochromatographic test strip has no cross-reaction with other foodborne pathogenic bacteria and has good specificity for the detection of TCBPE-stained Escherichia coli O157:H7.

[0067] Example 4 Determination of Simulated Bacterial Carriage in Food Samples by AIE Dual-Label Immunochromatographic Test Strip

[0068] Food samples simulating bacterial carriage were detected by the dual-label immunochromatographic test strip to verify the feasibility of the method based on TCBPE-labeled antigen and antibody of the test strip in the detection of food samples.

[0069] Beef, milk, and Chinese cabbage samples were purchased from the local market. 25 g or 25 mL of each sample was weighed and added to 225 mL of nutrient broth liquid medium. After homogenizing for 2 min, it was transferred to a conical flask and sterilized at 121 °C for 15 min. After cooling to room temperature, about 250 CFU of Escherichia coli O157:H7 was added respectively in a sterile environment, and then placed in an incubator at 37 °C for 12 h. At the same time, the immunochromatographic test strip was used to detect the culture broth collected every hour.

[0070] The results are as Figures 6 - 8 shown. For Chinese cabbage, milk, and beef sample media containing about 1 CFU / mL of Escherichia coli O157:H7, when the enrichment time was 1 - 3 h, only the quality control line of the dual-label immunochromatographic test strip had a green fluorescent band and there was no band at the test line; Figure 6 and 7 respectively showed that when the incubation time of Chinese cabbage and milk samples was 5 h, green fluorescent bands appeared at both the quality control line and the test line of the test strip, and the test results were positive; Figure 8 showed that when the enrichment time of beef samples was 4 h, a fluorescent band appeared at the test line of the test strip and a band also appeared at the quality control line, indicating that the test result was positive.

[0071] That is, when the TCBPE double-labeled immunochromatographic test strip is used to detect Chinese cabbage and milk samples contaminated with Escherichia coli O157:H7 at about 1 CFU / mL, Escherichia coli O157:H7 can be detected after 5 hours of enrichment. For beef samples contaminated with Escherichia coli O157:H7, Escherichia coli O157:H7 can be detected after 4 hours of enrichment. In addition, compared with the national standard method, the pre-enrichment time required by the present invention is shortened by more than 19 - 20 hours, greatly reducing the detection time and improving the detection speed.

[0072] In summary, it shows that the method of using the TCBPE double-labeled immunochromatographic test strip to detect Escherichia coli O157:H7 can be used for the highly sensitive and rapid detection of Escherichia coli O157:H7 in Chinese cabbage, milk and beef.

[0073] Based on the above experimental results, the advantages of the AIE double-labeled highly sensitive and rapid detection immunochromatographic test strip of the present invention are mainly as follows: (1) The detection sensitivity is 10 4 CFU / mL, which is 10 - 100 times higher than that of the conventional colloidal gold test strip, providing a guarantee for the accurate detection of Escherichia coli O157:H7; (2) The pre-enrichment time of the present invention only needs 4 - 5 hours, while the national standard method requires more than 24 - 36 hours. The detection time of the present invention is short, and the detection can be completed faster in application scenarios such as pollution source tracing, on-site detection and disease diagnosis, improving the timeliness of detection. By establishing this immunochromatographic test strip based on TCBPE-labeled antigen and antibody, it is expected to better realize commercial application in the highly sensitive and rapid detection of foodborne pathogenic bacteria.

[0074] The above-described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. An AIE dual-labeled immunochromatographic test strip for rapid and sensitive detection of Escherichia coli O157:H7, characterized in that: The immunochromatographic test strip is composed of a sample pad, a binding pad, a nitrocellulose membrane and an absorption pad which are sequentially connected and fixed on a bottom plate; The conjugate pad is coated with a TCBPE-labeled Escherichia coli O157:H7 detection antibody; The nitrocellulose membrane is coated with a detection line T and a quality control line C which are separated from each other in sequence; The detection line T is coated with Escherichia coli O157:H7 capture antibody; The quality control line C is coated with goat anti-mouse antibody.

2. The immunochromatographic test strip according to claim 1, wherein: The TCBPE-labeled Escherichia coli O157:H7 detection antibody includes Escherichia coli O157:H7 monoclonal antibody.

3. The immunochromatographic test strip according to claim 1, characterized in that: The method for preparing the TCBPE-labeled Escherichia coli O157:H7 detection antibody comprises the following steps: Adding TCBPE solution to boric acid buffer, then adding EDC solution and NHS solution to activate the carboxyl group of TCBPE, centrifuging to obtain the precipitate and resuspending to obtain the carboxyl group-activated TCBPE solution; The concentration of the E. coli O157:H7 detection antibody was adjusted to 1 mg / mL, 2 mL of carboxyl-activated TCBPE solution was added for labeling, and then a blocking solution was added for blocking, the precipitate was obtained by centrifugation, ultrasonic resuspending, and washing. The volume ratio of the boric acid buffer solution, the TCBPE solution, the EDC solution and the NHS solution is 20:1:2:1.

5.

4. The immunochromatographic test strip according to claim 1, wherein: The detection line T and the quality control line C are parallel to each other and have an equal width interval, and the interval is 0.5 cm.

5. The immunochromatographic test strip according to claim 1, characterized in that: The Escherichia coli O157:H7 capture antibody includes Escherichia coli O157:H7 monoclonal antibody.

6. The immunochromatographic test strip according to claim 1, characterized in that: The base plate comprises a PVC adhesive base plate.

7. Use of the immunochromatographic test strip as claimed in claim 1 in detecting Escherichia coli O157:H7.

8. A method for detecting Escherichia coli O157:H7, characterized in that: The method comprises the step of using the immunochromatographic test strip according to claim 1 to perform detection.

9. The method according to claim 8, characterized in that It also includes a sample pre-enrichment step.

10. The method according to claim 8, characterized in that The sample pre-enrichment comprises: adding the sample into a nutrient broth liquid culture medium, then adding TCBPE, and culturing for 4-5 hours.

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