Immunochromatography test strip for detecting shiga toxin-producing escherichia coli O103

Abstract

The invention relates to the technical field of analysis and detection, in particular to an immunochromatography test strip for detecting shiga toxin-producing escherichia coli O103. Based on the characteristics of the quantum dots and the fluorescent microspheres, a probe is synthesized by utilizing the purified protein expressed by the pET-28a-O103 strain and the fluorescent microspheres, and the immunochromatography rapid detection test strip for identifying the shiga toxin-producing escherichia coli O103, which is simple to operate, is established, can be used for rapidly identifying the shiga toxin-producing escherichia coli O103 clinically, and can be used for rapidly detecting the shiga toxin-producing escherichia coli O103. The method has important significance on prevention and control of Escherichia coli diseases.

Description

Technical Field

[0001] The invention relates to the technical field of analysis and detection, in particular to an immunochromatographic test strip for detecting Shiga toxin-producing Escherichia coli O103. Background Art

[0002] Escherichia coli (E coli), also known as Escherichia coli, is a Gram-negative, facultative anaerobic, rod-shaped bacterium of the genus Escherichia coli with blunt ends, motile, and non-spore-forming. Escherichia coli mainly exists in the intestines of humans and animals. Given the frequent interactions between animals, humans, and the environment, these bacteria often end up spreading to the external environment.

[0003] Human infection with E. coli is usually caused by eating contaminated food present in the food chain, especially animal-derived food. Pathogenic strains of E. coli can cause food poisoning, pneumonia, urinary tract infections, sepsis and meningitis and even death in humans, as well as similar diseases in animals. At least six pathological types associated with gastrointestinal infections have been discovered: enteropathogenic E. coli, enterotoxigenic E. coli, cytotoxin-producing E. coli or Shiga toxin-producing E. coli, enteroinvasive E. coli, enteroaggregative E. coli, and diffusely adherent E. coli.

[0004] Shiga toxin-producing Escherichia coli (STEC) is a type of Escherichia coli that can produce Shiga toxins. It is a Gram-negative short rod that is morphologically indistinguishable from common Escherichia coli. STEC are considered serious foodborne pathogens and they constitute a diverse group of organisms, which is related to their O antigen serogroups and virulence gene profiles. Globally, STEC is an important cause of diarrheal diseases, with symptoms including severe stomach cramps, bloody diarrhea, vomiting, etc. Severe infections can be life-threatening and lead to other complications. The most notable are hemorrhagic colitis and post-diarrhea sequelae such as hemolytic uremic syndrome (HUS). Cattle are the main reservoir of STEC and are transmitted to humans through consumption of contaminated food, fecal-oral transmission or cross-contamination. About half of human cases can be attributed to foodborne exposure.

[0005] STEC includes several pathogenic serogroups, the most common of which is STEC O157. Other STEC serotypes are collectively referred to as non-O157 STEC. Although STEC O157 is the main serotype responsible for many human STEC outbreaks, other serogroups are increasingly implicated in foodborne human illness cases. In recent years, the incidence of disease outbreaks associated with non-O157 STEC has increased, especially the first six non-O157 STEC, known as the "big six serogroups" O26, O45, O103, O111, O121, and O145. Studies have shown that STEC can be transmitted to humans through contaminated beef, milk, vegetables, fruits, and other foods, so it is critical to develop methods to detect all STEC to facilitate research on these pathogens. Currently, ISO methods are not suitable for the detection of non-O157 STEC, and there is no recognized standard method for the detection of non-O157 STEC in food.

[0006] Among non-O157 STEC, STEC O103 is of particular concern. Several studies have shown that STEC O103 is the second most common serotype causing human disease after O157. When animals are infected with STEC O103 serotype, they will develop symptoms similar to those of E. coli infection, which is difficult to distinguish based on clinical diagnosis alone.

[0007] At present, O157 and some non-O157 STEC are mainly identified by immunomagnetic separation, multiplex real-time polymerase chain reaction, real-time fluorescence quantitative PCR, lateral flow immunoassay, ELISA and other methods. However, the above methods all require expensive instruments and professional operators, and the detection time is long and not suitable for on-site diagnosis. Therefore, providing a method for detecting STEC O103 that can be fast, simple and accurate has become a technical problem that needs to be solved in this field. Summary of the invention

[0008] In order to solve the above technical problems, the present invention provides an immunochromatographic test strip, which comprises: a substrate and a sample pad, a conjugation pad, a reaction membrane and a water absorbent pad arranged on the substrate, wherein the sample pad, the conjugation pad, the reaction membrane and the water absorbent pad are overlapped in sequence; The reaction membrane is provided with a detection line and a quality control line spaced apart from each other, wherein the detection line is closer to the conjugate pad than the quality control line; the detection line (T line) is coated with a specific probe protein, and the quality control line (C line) is coated with rabbit anti-mouse IgG; the conjugate pad contains a complex of a specific probe protein and a labeled microsphere, and a complex of mouse IgG and a labeled microsphere; the specific probe protein is a protein expressed and purified by the pET-28a-O103 strain.

[0009] The immunochromatographic test strip can be used to quickly, simply and accurately identify STEC O103.

[0010] In some embodiments, the sample pad, conjugate pad, reaction membrane and absorbent pad overlap each other by 1 to 2 mm and are overlapped in sequence.

[0011] In some embodiments, the labeled microspheres are selected from fluorescent microspheres, colored microspheres, magnetic microspheres, or liposomes.

[0012] Preferably, the labeling microspheres are selected from quantum dot fluorescent microspheres, upconversion luminescent microspheres, colloidal gold, colloidal selenium or colloidal carbon.

[0013] Preferably, the labeled microspheres are quantum dot fluorescent microspheres.

[0014] Fluorescent microspheres, as functional microspheres embedded with quantum dots, have been widely used in the field of immunochromatography due to their luminescence properties, structural stability, and ease of modification. Fluorescent microsphere immunochromatography is an immunochromatography technique that uses fluorescent microspheres as markers. It has the advantages of being fast, simple, low-cost, and not requiring professional operators. It has been widely used in the field of clinical diagnosis in recent years. In addition, due to the fluorescent quantitative characteristics of fluorescent microspheres, this technology can achieve rapid qualitative and quantitative detection. Compared with colloidal gold immunochromatography, fluorescent microsphere immunochromatography exhibits higher sensitivity, providing a more accurate and efficient detection method for clinical diagnosis.

[0015] Based on the characteristics of quantum dots and fluorescent microspheres, the present invention uses the purified protein expressed by the pET-28a-O103 strain and the fluorescent microspheres to synthesize probes, and establishes a simple-to-operate immunochromatographic rapid test strip for detecting Shiga toxin-producing Escherichia coli O103. The test strip can quickly identify Shiga toxin-producing Escherichia coli O103 in clinical practice, which is of great significance for the prevention and control of Escherichia coli diseases.

[0016] In the specific implementation process, on the one hand, the present invention can be used in conjunction with an ultraviolet flashlight and can be widely used in farms to achieve differential diagnosis of Shiga toxin-producing Escherichia coli O103; on the other hand, it can be used in combination with a fluorescent immunoassay analyzer to achieve quantitative analysis and detection of Shiga toxin-producing Escherichia coli O103 in samples.

[0017] In some embodiments, the sample pad and the conjugate pad are each independently selected from glass fiber membrane, polyester fiber membrane, non-woven fabric, or a glass fiber and polyester mixed membrane.

[0018] In some embodiments, the reaction membrane is a nitrocellulose membrane; and / or the material of the substrate is selected from polyethylene, polyvinyl chloride, polypropylene or polystyrene.

[0019] In some embodiments, the complex of the specific probe protein and the labeled microspheres or the complex of mouse IgG and the labeled microspheres is prepared by coupling the specific probe protein or mouse IgG with the labeled microspheres using EDC and NHS.

[0020] Preferably, the preparation method of the complex of specific probe protein and labeled microspheres or the complex of mouse IgG and labeled microspheres comprises: mixing the labeled microspheres with MES buffer, then adding EDC and NHS for activation, centrifuging and resuspending the precipitate with MES buffer, and then mixing with the specific probe protein or mouse IgG, performing coupling reaction at room temperature in the dark, then adding BSA and glycine for blocking, and resuspending the precipitate with a re-solution after centrifugation; the re-solution comprises 0.05~0.2 mol / L pH 7.5~8.5 TBST buffer, 5%~10% (w / v) sucrose, 1%~4% (w / v) D-trehalose, 0.5%~2% (w / v) BSA, and 0.1%~1% (w / v) sodium caseinate.

[0021] Preferably, the conjugate pad is first soaked and dried with a conjugate pad treatment solution, and then sprayed with a complex of a specific probe protein and labeled microspheres, and a complex of mouse IgG and labeled microspheres; the conjugate pad treatment solution includes 0.05~0.2 mol / L pH 7.2~7.6 PBS buffer, 1%~5% (w / v) sucrose, 1%~3% (w / v) BSA and 1%~3% (w / v) Tween-20.

[0022] Furthermore, the present invention provides a kit containing the above-mentioned immunochromatographic test strip.

[0023] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a rapid, simple and accurate immunochromatographic test strip for detecting Shiga toxin-producing Escherichia coli O103, which can perform differential diagnosis on Shiga toxin-producing Escherichia coli O103 and has the characteristics of strong specificity, high sensitivity, short detection time, simple operation, etc. The present invention can provide a rapid differential diagnosis detection method for Shiga toxin-producing Escherichia coli O103 in aquaculture and food safety, reduce detection time and cost, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a schematic diagram of a quantum dot fluorescent microsphere immunochromatographic rapid test strip for identifying Shiga toxin-producing Escherichia coli O103.

[0025] Figure 2 It is a reference chart for the test results of the immunochromatographic rapid test strips.

[0026] Figure 3 It is a sensitivity test result diagram.

[0027] Figure 4 It is a graph of specificity test results.

[0028] Figure 5 This is a graph of clinical sample testing results. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] In the examples provided in this specification, if no specific techniques or conditions are specified, the techniques or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0031] The synthesis method of the fluorescent microspheres in the following examples is as follows, and the reagents are all from Sigma: 68 mg of triethanolamine was added to 25 ml of water, heated and stirred at 80 degrees for half an hour, and then 380 mg of CTAB and 168 mg of sodium salicylate were added. After stirring for 1 hour, 4 ml of TEOS solution was added and the reaction was continued at 80 degrees for 2 hours. Then, the precipitate was collected after dilution with anhydrous ethanol, washed three times with ethanol, dispersed in a mixed solution of methanol and hydrochloric acid, refluxed at 60 degrees for 6 hours, and repeated once, and the precipitate was collected to obtain dendritic silica nanospheres.

[0032] The above precipitate was dispersed in 100 ml of anhydrous ethanol, and 2.5 ml of ammonia water and 1 ml of MPTS were added to the above solution. The mixture was stirred vigorously at room temperature for 12 hours, and the precipitate was collected by centrifugation, washed three times with anhydrous ethanol, and dispersed in 50 ml of anhydrous ethanol to obtain thiol-modified dendritic silica spheres. Take 1ml of the above ethanol dispersion, add 500ul QDs (Suzhou Xingshuo Nano Co., Ltd.), mix ultrasonically for 5min, centrifuge to remove excess QDs. The precipitate is dispersed in the PVP solution, ultrasonically treated for 20min, centrifuged, and the obtained precipitate is dispersed in a mixed solution containing 40ml anhydrous ethanol, 10ml water, and 1ml ammonia water. 30ul TEOS is added, stirred at room temperature for 3 hours, centrifuged, washed three times with ethanol, and the obtained precipitate is dispersed in a mixed solution containing 40ml ethanol, 1ml ammonia water, and 40ul APTS, and reacted at room temperature for 12 hours. The above product is centrifuged, washed three times with ethanol, dispersed in 20ml DMF solution, 200mg succinic anhydride is added, and reacted for 6 hours. The above product is centrifuged, washed three times with ethanol, and dispersed in an ethanol solution for subsequent biofunctionalization modification.

[0033] The brand and product number of mouse IgG in the following examples are Proteintech B900620; the brand and product number of rabbit anti-mouse IgG are Proteintech B900120.

[0034] The pET-28a-0103 strain in the following examples is disclosed in steps 1 and 2 of Example 1 of CN 116903710 A. The protein expressed and purified by the pET-28a-0103 strain was obtained according to the following expression and purification method.

[0035] pET-28a-0103 was coated on a plate containing kanamycin (Kana + ) LB solid plate, pick a single colony to 5ml LB culture medium (Kana + ) for overnight culture, and then transferred into 500 ml LB culture medium (Kana + ) Waiting for OD 600 nm When the value reached 0.6, PTG was added at a final concentration of 1 mmol / L for overnight induction of expression. The bacterial solution was then collected and centrifuged to discard the supernatant. The bacterial precipitate was added with buffer and ultrasonically broken. The supernatant was used for purification. The purification steps followed the "balance-loading-washing-elution" method. The eluate was collected for dialyzation and concentration, and the protein concentration was determined by the BCA method.

[0036] Example 1 This embodiment provides a quantum dot fluorescent microsphere immunochromatographic rapid test strip for identifying Shiga toxin-producing Escherichia coli O103. The immunochromatographic test strip consists of a sample pad, a binding pad, a nitrocellulose membrane (reaction membrane), a water-absorbing pad, and a PVC bottom plate (substrate). The nitrocellulose membrane is used as the detection membrane of the test strip, and a detection line (T line) coated with a protein expressed and purified by the pET-28a-O103 strain is drawn near the left end, and a quality control line (C line) coated with rabbit anti-mouse IgG is drawn at a position 5 mm apart from the right end of the detection line (T line). An absorbent pad is attached to the right end of the nitrocellulose membrane. A binding pad is provided at the left end of the nitrocellulose membrane, and the binding pad is made of a glass fiber membrane, on which a mixture of a protein-fluorescent microsphere conjugate expressed and purified by the pET-28a-O103 strain and a mouse IgG-fluorescent microsphere conjugate is sprayed. A sample pad is provided at the left end of the binding pad. The sample pad, conjugate pad, nitrocellulose membrane and absorbent pad are overlapped on the PVC base plate in sequence, and each part overlaps 1-2 mm to ensure that the test strip can smoothly complete the entire flow process after the sample is added. Cut it into 4 mm wide test strips and put it into a plastic card shell to form a complete test strip.

[0037] The detection principle and result judgment method of the above-mentioned immunochromatographic test strip are as follows: When the test sample contains Shiga toxin-producing Escherichia coli O103, it will bind to the protein-fluorescent microsphere conjugate expressed and purified by the pET-28a-O103 strain on the binding pad to form a "fluorescent microsphere-protein-O103" complex. The complex moves toward the absorbent pad under the action of capillaries. When it flows to the detection line of the nitrocellulose membrane, the protein-fluorescent microsphere-protein-O103 expressed and purified by the pET-28a-O103 strain coated on the detection line will be absorbed by the complex. The white is captured to form a "fluorescent microsphere-protein-O103-protein" double antigen sandwich complex, that is, the test line can be seen under ultraviolet light, and the excess pET-28a-O103 strain expressed purified protein-fluorescent microsphere conjugate and mouse IgG-fluorescent microsphere conjugate continue to flow forward. When it flows to the nitrocellulose membrane quality control line, it reacts with the rabbit anti-mouse IgG coated on the quality control line to form a "fluorescent microsphere-mouse IgG-rabbit anti-mouse IgG" complex, that is, under ultraviolet light, a red fluorescent quality control line can be seen. If there is no Shiga toxin-producing Escherichia coli O103 in the sample, the test line cannot capture the fluorescent microsphere complex, and the test line will not appear fluorescent strips, but the "fluorescent microsphere-mouse IgG-rabbit anti-mouse IgG" complex will still form on the quality control line to appear fluorescent strips. Regardless of whether there are Shiga toxin-producing Escherichia coli O103 in the sample, the quality control line should have fluorescent strips. If no fluorescent strips appear on the quality control line, the test result is invalid.

[0038] The preparation method of the above-mentioned immunochromatographic test strip is as follows: 1. Preparation of fluorescent microsphere-labeled antibody conjugates Prepare labeling buffer: 0.1 mol / L pH 6 MES buffer Prepare the reconstitution solution: 0.1 mol / L pH 8 TBST buffer, 8% (w / v) sucrose, 2% (w / v) D-trehalose, 1% (w / v) BSA, and 0.5% (w / v) caseinate sodium.

[0039] The specific method for preparing the protein-fluorescent microsphere conjugate and mouse IgG-fluorescent microsphere conjugate expressed and purified by the pET-28a-O103 strain includes the following: In the first step, 10 μL of fluorescent microspheres were mixed with 990 μL of labeling buffer, and then 10 μL of 10 mg / mL EDC labeling activation solution and 10 μL of 50 mg / mL suflo-NHS labeling stabilization solution were added. The mixture was reacted at room temperature in the dark for 30 min to activate the carboxyl groups on the surface of the fluorescent microspheres.

[0040] In the second step, after activation, centrifuge at 12,000 rpm for 30 min at 4 °C, discard the supernatant, resuspend the precipitate with 1 mL of labeling buffer, add 5 μg of pET-28a-O103 strain expressed purified protein and 8 μg of mouse IgG, respectively, and react at room temperature in the dark for 6 h.

[0041] In the third step, 10 μL of 10% (w / v) BSA was added to block the reaction for 1 h in the dark, and then 100 mg of glycine was added to continue the blocking reaction for 2 h.

[0042] Step 4. After blocking, centrifuge at 12,000 rpm at 4 °C for 20 min, discard the supernatant, resuspend with 1 mL of resuspending solution, and store at 4 °C in the dark.

[0043] 2. Preparation of fluorescent microsphere conjugate pad Prepare the conjugate pad treatment solution: 0.1 mol / L pH 7.4 PBS buffer, 3% (w / v) sucrose, 2% (w / v) BSA, and 2% (w / v) Tween-20.

[0044] The specific method for preparing the fluorescent microsphere conjugate pad includes the following: Cut the glass fiber membrane into 1 cm × 30 cm strips, soak them evenly with the above-mentioned treatment solution, and then place them in a 37 ℃ environment to dry for 8-12 h; mix the protein-fluorescent microsphere conjugate expressed and purified by the pET-28a-O103 strain prepared in step 1 and the mouse IgG-fluorescent microsphere conjugate evenly, then spray them onto the treated binding pad at a spray volume of 6 μL / cm, and dry them in a 37 ℃ incubator for use.

[0045] 3. Spraying inspection line and quality control line The specific methods of spraying inspection line and quality control line include the following: The purified protein expressed by the pET-28a-O103 strain and rabbit anti-mouse IgG were diluted with PBS buffer and sprayed on the nitrocellulose membrane as the detection line (T line) and quality control line (C line), respectively. The concentration of the purified protein expressed by the pET-28a-O103 strain was 0.2 mg / mL, and the spray volume was 1 μL / cm; the concentration of the rabbit anti-mouse IgG sprayed on the nitrocellulose membrane was 0.5 mg / mL, and the spray volume was 1 μL / cm. The interval between the detection line (T line) and the quality control line (C line) was 5 mm, and the samples were dried at 37 °C for use.

[0046] 4. Assembly of test strips The specific method of assembling the test strips includes the following: The processed sample pad, binding pad, nitrocellulose membrane and absorbent pad are pasted to the PVC base plate in order, with each part overlapping by 1-2 mm, and then cut into test strips with a width of 4 mm and put into a plastic card shell to form a complete test strip, which is then placed in a clean aluminum foil bag, added with a desiccant, sealed and stored in a constant temperature and humidity storage cabinet away from light for future use. The schematic diagram of the quantum dot fluorescent microsphere immunochromatographic rapid detection test strip for identifying Shiga toxin-producing Escherichia coli O103 in this embodiment is shown in FIG. Figure 1 As shown in the figure, the test results are shown in Figure 2 shown.

[0047] Example 2 In this example, the performance of the immunochromatographic test strip prepared in Example 1 was tested.

[0048] 1. Sensitivity detection Shiga toxin-producing Escherichia coli O103 was diluted with PBS buffer to obtain Shiga toxin-producing Escherichia coli O103 reference samples with different dilution multiples and negative samples. Then 80 μL of the sample was added to the sample pad of the test strip. After 10 minutes, the test results were observed with an ultraviolet flashlight, and the fluorescence intensity on the test line was read using a dry fluorescent immunoassay analyzer.

[0049] Test results such as Figure 3 As shown, the diluted Shiga toxin-producing Escherichia coli O103 reference (10 2 The test strip of Example 1 was still positive after 3 repetitions, and the results were the same, indicating that the test strip of Example 1 was highly sensitive.

[0050] 2. Specificity detection The test strips of Example 1 were used to detect positive references of Escherichia coli O157, O9, O21, O88, O103, O111, Merril anatipestifer, Staphylococcus aureus, Salmonella, and Shiga toxin-producing Escherichia coli O103. 80 μL of the sample was diluted with PBS buffer and dripped onto the sample pad of the test strip.

[0051] Test results such as Figure 4 As shown, when testing the positive reference of Shiga toxin-producing Escherichia coli O103, the test line appeared; when testing other virus positive references, the test line was negative, there was no cross reaction, and the results were the same after three repetitions, indicating that the test strip has a high specificity.

[0052] 3. Clinical sample testing Several clinical positive and negative samples of Shiga toxin-producing Escherichia coli O103 with known background were extracted and diluted in PBS buffer. The clinical samples were tested by PCR using bacterial universal primers and the fluorescent microsphere immunochromatographic test strip of Example 1 were used for detection at the same time. The detection results of the two methods were compared.

[0053] The test results are as follows Figure 5 As shown, when testing positive clinical samples with different concentrations of Shiga toxin-producing Escherichia coli O103, the test line appears; when testing negative samples, the test line is negative, indicating that the test strip has the ability to detect clinical samples.

[0054] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An immunochromatographic test strip, characterized in that: The immunochromatographic test strip comprises: a substrate and a sample pad, a conjugation pad, a reaction membrane and a water absorbent pad arranged on the substrate, wherein the sample pad, the conjugation pad, the reaction membrane and the water absorbent pad are overlapped in sequence; The reaction membrane is provided with a detection line and a quality control line spaced apart from each other, the detection line is closer to the binding pad than the quality control line; the detection line is coated with a specific probe protein, and the quality control line is coated with rabbit anti-mouse IgG; the binding pad contains a complex of a specific probe protein and a labeled microsphere, and a complex of mouse IgG and a labeled microsphere; the specific probe protein is a protein expressed and purified by the pET-28a-O103 strain.

2. The immunochromatographic test strip according to claim 1, characterized in that: The sample pad, conjugate pad, reaction membrane and water absorbent pad overlap each other by 1 to 2 mm and are overlapped in sequence.

3. The immunochromatographic test strip according to claim 1, characterized in that: The labeled microspheres are selected from fluorescent microspheres, colored microspheres, magnetic microspheres or liposomes.

4. The immunochromatographic test strip according to claim 1, characterized in that: The labeling microspheres are selected from quantum dot fluorescent microspheres, up-conversion luminescent microspheres, colloidal gold, colloidal selenium or colloidal carbon.

5. The immunochromatographic test strip according to claim 1, characterized in that: The sample pad and the conjugate pad are independently selected from glass fiber membrane, polyester fiber membrane, non-woven fabric or glass fiber and polyester mixed membrane.

6. The immunochromatographic test strip according to claim 1, characterized in that: The reaction membrane is a nitrocellulose membrane; and / or the material of the substrate is selected from polyethylene, polyvinyl chloride, polypropylene or polystyrene.

7. The immunochromatographic test strip according to claim 1, characterized in that: The complex of the specific probe protein and the labeled microspheres or the complex of the mouse IgG and the labeled microspheres is prepared by coupling the specific probe protein or the mouse IgG with the labeled microspheres using EDC and NHS.

8. The immunochromatographic test strip according to claim 7, characterized in that: The preparation method of the complex of specific probe protein and labeled microspheres or the complex of mouse IgG and labeled microspheres includes: mixing the labeled microspheres with MES buffer, then adding EDC and NHS for activation, centrifuging and resuspending the precipitate with MES buffer, mixing with the specific probe protein or mouse IgG, performing coupling reaction at room temperature in the dark, then adding BSA and glycine for blocking, centrifuging and resuspending the precipitate with a re-solution; the re-solution includes 0.05~0.2 mol / L pH 7.5~8.5 TBST buffer, 5%~10% (w / v) sucrose, 1%~4% (w / v) D-trehalose, 0.5%~2% (w / v) BSA, and 0.1%~1% (w / v) casein sodium.

9. The immunochromatographic test strip according to claim 8, characterized in that: The conjugate pad is first soaked and dried with a conjugate pad treatment solution, and then sprayed with a complex of a specific probe protein and a labeled microsphere, and a complex of mouse IgG and a labeled microsphere; the conjugate pad treatment solution includes 0.05-0.2 mol / L pH 7.2-7.6 PBS buffer, 1%-5% (w / v) sucrose, 1%-3% (w / v) BSA, and 1%-3% (w / v) Tween-20.

10. A kit for detecting Shiga toxin-producing Escherichia coli O103, characterized in that: It contains the immunochromatographic test strip according to any one of claims 1 to 9.

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