ELISA kit and method for detecting vibrio vulnificus

Abstract

The invention discloses an ELISA (Enzyme Linked Immunosorbent Assay) kit for detecting vibrio vulnificus and a method thereof, and aims to provide the ELISA kit which is rapid, sensitive, simple to operate and capable of detecting the vibrio vulnificus in a high-throughput manner. According to the technical scheme, the kit comprises a coating antigen, a sealing agent, a platinum-palladium-rhodium nanocomposite-labeled vibrio vulnificus rabbit-derived polyclonal antibody, a PBST washing plate, an elisa plate, a PBS buffer solution, a stop solution and a TMB developing solution.

Description

Technical Field

[0001] The present application belongs to the technical field of Vibrio vulnificus detection, and in particular relates to the establishment of a direct ELISA method for detecting Vibrio vulnificus. Background Art

[0002] Vibrio vulnificus belongs to the Vibrio family and is a Gram-negative bacterium. It is one of the three major pathogenic Vibrio species that endanger human health, along with Vibrio cholerae and Vibrio parahaemolyticus. It is known as "flesh-eating bacteria" and is one of the pathogenic Vibrio species with the highest mortality rate. It is widely distributed in the waters at the junction of rivers and seas, offshore, bays and seabed sediments in the world with high water temperatures, and often parasitizes in shellfish (such as oysters, clams, etc.). Vibrio vulnificus can infect seafood, and humans are mainly infected by eating uncooked contaminated seafood or by wounds coming into contact with seawater.

[0003] Patients infected by ingestion quickly develop gastroenteritis-type symptoms, and the mortality rate of patients infected by ingestion who further develop sepsis is as high as over 50%. In the United States and coastal areas, it is the main cause of seafood-related deaths and diseases, accounting for about 90% of deaths caused by Vibrio in these areas. The mortality rate of Vibrio vulnificus infection is extremely high, about 25% in wound infection cases, about 50% in sepsis cases, and about 33% in gastroenteritis cases, even with treatment. The pathogenicity of Vibrio vulnificus stems from the immune escape of the polysaccharide capsule, the inflammatory response caused by endotoxins, the destruction of cell tissues by exotoxins such as MARTX toxins, and the efficient iron uptake ability. Vibrio vulnificus outbreaks are mostly concentrated in the summer, in warm coastal areas such as the southeastern United States, the Gulf of Mexico, Asia, the Middle East, and Europe.

[0004] Currently, the diagnosis of Vibrio vulnificus involves a variety of technical means, including traditional culture and biochemical identification, serological testing, immunoassays based on antigen-antibody recognition, and modern molecular methods such as PCR. Traditional detection methods are the main means of clinical diagnosis, mainly including morphological detection, culture separation, and biochemical detection.

[0005] However, traditional detection methods are time-consuming, and there are many types of pathogens of infectious diseases. Only a few common pathogens can be detected, and the detection items rely largely on the judgment of clinicians, which can no longer meet clinical needs. ELISA detection has the advantages of high sensitivity, strong specificity, quantitative detection, wide application range and good repeatability. Therefore, it is of great significance to develop a rapid, sensitive, accurate and high-throughput ELISA detection method for Vibrio vulnificus in seawater and seafood. Summary of the invention

[0006] Based on this, the first object of the present invention is to provide an ELISA kit for rapid, sensitive, simple to operate and high-throughput detection of Vibrio vulnificus.

[0007] The second purpose of the present invention is to provide the application of the above kit.

[0008] The third object of the present invention is to provide a method for detecting Vibrio vulnificus using an ELISA kit.

[0009] To this end, the first technical solution provided by the present invention is as follows:

[0010] An ELISA kit for detecting Vibrio vulnificus comprises a coating antigen, a blocking agent, a Vibrio vulnificus rabbit-derived polyclonal antibody labeled with a platinum-palladium-rhodium nanocomposite, a PBST plate washer, an ELISA plate, a PBS buffer, a stop solution, and a TMB color developing solution.

[0011] Furthermore, in the above-mentioned ELISA kit for detecting Vibrio vulnificus, the blocking agent is skimmed milk powder.

[0012] Furthermore, in the above-mentioned ELISA kit for detecting Vibrio vulnificus, the Vibrio vulnificus rabbit-derived polyclonal antibody is prepared by the following method: inactivated Vibrio vulnificus is mixed with Freund's adjuvant to prepare a vaccine, the vaccine is used to immunize experimental rabbits at intervals, the serum of the experimental rabbits is collected, and the Vibrio vulnificus polyclonal antibody is extracted and purified from the serum to obtain the Vibrio vulnificus polyclonal antibody.

[0013] Furthermore, in the above-mentioned ELISA kit for detecting Vibrio vulnificus, the method for preparing rabbit-derived polyclonal antibodies to Vibrio vulnificus labeled with platinum, palladium, and rhodium nanocomplexes is as follows: adding rabbit-derived polyclonal antibodies to Vibrio vulnificus to a dispersion of palladium and rhodium nanocomplexes for reaction, then adding BSA blocking solution, and finally centrifuging to remove the supernatant to obtain rabbit-derived polyclonal antibodies to Vibrio vulnificus labeled with platinum, palladium, and rhodium nanocomplexes.

[0014] Furthermore, in the above-mentioned ELISA kit for detecting Vibrio vulnificus, the platinum, palladium, and rhodium nanocomplex is prepared by the following method: H2PtCl6, Na2PdCl4, RhCl3, and HCl are mixed, poloxamer 407 (F127) is added, and then ultrasonically dissolved, and then L-ascorbic acid is added, and the reaction is carried out at 95°C for 4 hours; after centrifugation, the precipitate is washed with water for multiple times to prepare a platinum, palladium, and rhodium nanocomplex.

[0015] Furthermore, in the above-mentioned ELISA kit for detecting Vibrio vulnificus, the method for preparing rabbit-derived polyclonal antibodies to Vibrio vulnificus labeled with platinum, palladium, and rhodium nanocomplexes is as follows: adding rabbit-derived polyclonal antibodies to Vibrio vulnificus to a dispersion of palladium and rhodium nanocomplexes for reaction, then adding BSA blocking solution, and finally centrifuging to remove the supernatant to obtain rabbit-derived polyclonal antibodies to Vibrio vulnificus labeled with platinum, palladium, and rhodium nanocomplexes.

[0016] Furthermore, in the above-mentioned ELIS / A kit for detecting Vibrio vulnificus, the TMB color developing solution formula is: 0.5 mL of ethanol solution containing 0.2% w / v 3,3',5,5'-tetramethylbenzidine, 4.5 mL of pure water, 5 mL of phosphate citrate buffer, and 10 μL of 30% w / w hydrogen peroxide.

[0017] Furthermore, in the above-mentioned ELISA kit for detecting Vibrio vulnificus, the stop solution is 2M sulfuric acid.

[0018] The second technical solution provided by the present invention is as follows:

[0019] A method for detecting Vibrio vulnificus, comprising: using the ELISA kit for detecting Vibrio vulnificus according to claim 1 for detection.

[0020] The third technical solution provided by the present invention is a method for detecting Vibrio vulnificus, which comprises the following steps in sequence:

[0021] (1) Add 100 μL of 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 CFU / mL of Vibrio vulnificus, incubate at 37°C for 2 h, then wash the plate with PBST and pat dry;

[0022] (2) Each well was blocked with 200 μL of 3 w / v% skim milk powder at 37°C for 1 h, then the plate was washed with PBST and patted dry;

[0023] (3) diluting the Vibrio vulnificus rabbit polyclonal antibody labeled with platinum-palladium-rhodium nanocomplex by 80 times with PBS buffer to obtain a probe diluent, adding 100 μL of the probe diluent to each well of the ELISA plate obtained in step (2), and then incubating the ELISA plate at 37° C. for 1 h, then washing the plate with PBST and patting dry;

[0024] (4) Add 100 μL of colorimetric solution to each well of the ELISA plate obtained in step (3), place the plate at 37°C for 15 min, then add 50 μL of 2M H2SO4 stop solution to each well, and measure the absorbance of the solution at 450 nm using an ELISA reader. According to the P / N value, when the catalytic substrate produces an absorbance value P / N ≥ 2.1 at 450 nm, it is judged as positive; when P / N < 2.1, it is judged as negative.

[0025] Furthermore, in the above-mentioned method for detecting Vibrio vulnificus, the inactivated Vibrio vulnificus is prepared by the following method: resuscitating the Vibrio vulnificus strain and inoculating it into a 3% sodium chloride alkaline water peptone water liquid culture medium for amplification culture, washing the amplified Vibrio vulnificus by centrifugation with PBS, removing the liquid culture medium, and obtaining a Vibrio vulnificus precipitate; finally, resuspending the Vibrio vulnificus precipitate with PBS and adding 1v / v% formaldehyde solution for inactivation culture, washing the Vibrio vulnificus bacterial liquid by centrifugation with PBS, removing the formaldehyde solution, and coating the obtained Vibrio vulnificus bacterial liquid on a nutrient agar plate, and obtaining an inactivated Vibrio vulnificus when no colonies grow.

[0026] Compared with the prior art, the technical solution provided by the present invention has the following advantages:

[0027] 1. The Vibrio vulnificus antibody prepared by the present invention has high specificity and has no cross reaction with other bacteria;

[0028] 2. The ELISA kit for detecting Vibrio vulnificus based on platinum palladium rhodium nanozyme of the present invention is simple to operate, low in cost, and highly reliable when detecting Vibrio vulnificus. It can quickly and accurately detect Vibrio vulnificus and can simultaneously detect a large number of samples. It is suitable for large-scale, high-throughput rapid detection requirements of food and clinical samples, has high economic and social value, and is worthy of promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art.

[0030] Figure 1 A schematic diagram of the structure of a direct ELISA method for detecting Vibrio vulnificus provided in an embodiment of the present application;

[0031] Figure 2 Transmission electron microscopy image and particle size distribution diagram (a), and scanning electron microscopy image (b) of the platinum palladium rhodium nanozyme provided in the examples of the present application;

[0032] Figure 3 To verify the peroxidase-like activity of PtPdR nanozymes;

[0033] Figure 4 To optimize the blocking conditions of the direct ELISA method based on PtPdRh nanozyme to detect Vibrio vulnificus, (a) zate potential of PtPdRh enzyme and PtPdRh nanobioprobe, (b) reaction system; (c) blocking agent; (d) blocking time;

[0034] Figure 5 This is the standard curve of the direct ELISA method for detecting Vibrio vulnificus based on the PtPdR nanobioprobe;

[0035] Figure 6 This is an experiment to test the specificity of the direct ELISA method for detecting Vibrio vulnificus based on platinum-palladium-rhodium nanobioprobe. DETAILED DESCRIPTION

[0036] The present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. In the description of the present invention, it should be noted that, if no specific conditions are specified in the embodiments, the conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0037] Example 1

[0038] The present embodiment provides an ELISA kit for detecting Vibrio vulnificus, comprising a coating antigen, a blocking agent, a Vibrio vulnificus rabbit-derived polyclonal antibody labeled with a platinum-palladium-rhodium nanocomposite, a PBST plate washer, an ELISA plate, a PBS buffer, a stop solution, and a TMB colorimetric solution.

[0039] The Vibrio vulnificus rabbit-derived polyclonal antibody labeled with platinum-palladium-rhodium nanocomposite is prepared by the following method:

[0040] 1) Preparation of platinum, palladium, and rhodium nanocomposites: 2.0 mL H2PtCl6 (20 mM), 1.0 mL Na2PdCl4 (20 mM), and 1.5 mL RhCl3 (20 mM) were successively added to a flask, 0.2 mL HCl (6 M) was added to the flask and shaken well, and then 50 mg poloxamer 407 (F127, Meilun Biotechnology Co., Ltd.) was added and dissolved by ultrasonic to form a uniform solution; 2.0 mL of freshly prepared L-ascorbic acid (0.1 M) was added to the above solution under magnetic stirring, the oil bath was heated to 95°C and maintained for 4 h, the solution was cooled to room temperature, the precipitate was collected by centrifugation, and washed three times with water, and the obtained nanoparticles were dispersed in deionized water.

[0041] The morphology and particle size of PtPdR nanozymes are as follows Figure 2 As shown, the platinum, palladium, and rhodium nanoparticles have good dispersibility in aqueous solution, with an average diameter of 93 nm and a rough porous structure on the surface.

[0042] 2) Preparation of rabbit-derived polyclonal antibodies against Vibrio vulnificus, specifically according to the following method:

[0043] 2.1) resuscitating the Vibrio vulnificus strain and inoculating it into a 3% sodium chloride alkaline peptone water liquid culture medium for amplification culture, washing the amplified Vibrio vulnificus with PBS by centrifugation, removing the liquid culture medium, and obtaining a Vibrio vulnificus precipitate; finally, resuspending the Vibrio vulnificus precipitate with PBS and adding 1v / v% formaldehyde solution for inactivation culture, washing the Vibrio vulnificus liquid with PBS by centrifugation, removing the formaldehyde solution, and coating the obtained Vibrio vulnificus liquid on a nutrient agar plate, and obtaining an inactivated Vibrio vulnificus when no colonies grow;

[0044] Vibrio vulnificus was activated and expanded by 3 wt% sodium chloride alkaline peptone water (Qingdao Hi-Tech Industrial Park Haibo Biotechnology Co., Ltd.). After culture, round, flat, opaque in the center, transparent at the edge, characteristic yellow colonies appeared. The Vibrio vulnificus strain was expanded and cultured, and the resulting bacterial solution was inactivated with 1% (v / v) formaldehyde for 24 h, and its concentration was adjusted to 10 9 CFU / mL. After the inactivated bacterial solution is cultured on a plate and no bacteria grow, the prepared Vibrio vulnificus is used as the whole-cell antigen of Vibrio vulnificus, which is packaged and stored at 4°C for later use.

[0045] The prepared whole bacterial antigen was used to immunize New Zealand white rabbits. Before the first immunization, 0.5 mL of Freund's complete adjuvant was mixed with 0.5 mL of inactivated Vibrio vulnificus and emulsified, and then the New Zealand white rabbits were subcutaneously injected at multiple points. After 20 days, the booster immunization was performed. 0.5 mL of Freund's incomplete adjuvant was mixed with 0.5 mL of inactivated Vibrio vulnificus and emulsified, and then subcutaneously injected at multiple points in the New Zealand white rabbits. Seven days later, blood was collected from the ear vein of the white rabbits, and the antibody titer was determined by indirect ELISA. If the titer was not ideal, the booster immunization was continued. The interval between each immunization was 14 days, and blood was collected seven days after each immunization to detect the titer. The total number of immunizations did not exceed six times to avoid tolerance in the white rabbits. If the titer reached the expected value, a large amount of blood could be taken from the white rabbits, and the serum was separated by centrifugation. After purification by saturated ammonium sulfate salt precipitation method, it was stored in a -20°C refrigerator.

[0046] 2.2) H2PtCl6, Na2PdCl4, RhCl3 and HCl were mixed, F127 was added and dissolved by ultrasonic, and then L-ascorbic acid was added and reacted at 95°C for 4h; after centrifugation, the precipitate was washed with water for multiple times to prepare a platinum-palladium-rhodium nanocomposite.

[0047] 2.3) Adding the Vibrio vulnificus rabbit polyclonal antibody to the Pd / Rh nanocomposite dispersion to react, then adding BSA blocking solution, and finally centrifuging to remove the supernatant to obtain the Vibrio vulnificus rabbit polyclonal antibody labeled with Pt / Pd / Rh nanocomposite.

[0048] Example 2

[0049] A method for detecting Vibrio vulnificus comprises the following steps in sequence:

[0050] (1) Add 100 μL of 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 CFU / mL of Vibrio vulnificus, incubate at 37°C for 2 h, then wash the plate with PBST and pat dry;

[0051] (2) Each well was blocked with 200 μL of 3 w / v% skim milk powder at 37°C for 1 h, then the plate was washed with PBST and patted dry;

[0052] (3) diluting the Vibrio vulnificus rabbit polyclonal antibody labeled with platinum-palladium-rhodium nanocomplex by 80 times with PBS buffer to obtain a probe diluent, adding 100 μL of the probe diluent to each well of the ELISA plate obtained in step (2), and then incubating the ELISA plate at 37° C. for 1 h, then washing the plate with PBST and patting dry;

[0053] (4) Add 100 μL of colorimetric solution to each well of the ELISA plate obtained in step (3), place the plate at 37°C for 15 min, then add 50 μL of 2M H2SO4 stop solution to each well, and measure the absorbance of the solution at 450 nm using an ELISA reader. According to the P / N value, when the catalytic substrate produces an absorbance value P / N ≥ 2.1 at 450 nm, it is judged as positive; when P / N < 2.1, it is judged as negative.

[0054] In order to demonstrate the technical advantages of the technical solution provided by the present invention, the following is a research experiment on the technical solution provided by the present application:

[0055] 1. Optimization of the amount of platinum-palladium-rhodium probe antibody:

[0056] Take 1mL of 0.1mg / mL PtPdRh nanozyme solution, add 2, 4, 6, 8, 10, 12μL of 1mg / mL purified Vibrio vulnificus polyclonal antibody, and stir the mixture at room temperature for 1h. Add 100μL of 10% (w / v) BSA solution and stir at room temperature for 1h to block the remaining sites. After the mixture is centrifuged (4℃, 6500rpm, 10min), the precipitate is re-dissolved with 50μL of re-solution (PtPdRh@pAb probe).

[0057] (1) Add 100 μL of 10 8 CFU / mL of Vibrio vulnificus were incubated at 37°C for 2 h, then the plates were washed with PBST and patted dry;

[0058] (2) Each well was blocked with 200 μL of 3% (w / v) skim milk powder at 37°C for 1 h, then the plate was washed with PBST and patted dry;

[0059] (3) After diluting the PtPdRh@pAb probe prepared above by 80 times, 100 μL of probe dilution solution was added to each well, and the ELISA plate was incubated at 37°C for 1 h, then washed with PBST and patted dry;

[0060] (4) Add 100 μL of colorimetric solution to each well, place the plate at 37°C for 15 min, then add 50 μL of 2 M H2SO4 stop solution to each well and measure the absorbance of the solution at 450 nm using an ELISA reader.

[0061] 2. Optimization of Pt / Pd / Rh probe coupling pH:

[0062] Take 1mL of 0.1mg / mL PtPdRh nanozyme solution, add 2, 4, 6, 8μL of 0.1M HCl and 2, 4, 6, 8μL of 0.2M K2CO3, respectively, and stir for 5min. Then add 8μL of 1mg / mL purified anti-V. vulnificus polyclonal antibody, and stir the mixture at room temperature for 1h. Add 100μL of 10% (w / v) BSA solution and stir at room temperature for 1h to block the remaining sites. After the mixture is centrifuged (4℃, 6500rpm, 10min), the precipitate is re-dissolved with 50μL of re-solution.

[0063] (1) Add 100 μL of 10 8 CFU / mL of Vibrio vulnificus were incubated at 37°C for 2 h, then the plates were washed with PBST and patted dry;

[0064] (2) Each well was blocked with 200 μL of 3% (w / v) skim milk powder at 37°C for 1 h, then the plate was washed with PBST and patted dry;

[0065] (3) After diluting the PtPdRh@pAb probe prepared above by 80 times, 100 μL of probe dilution solution was added to each well, and the ELISA plate was incubated at 37°C for 1 h, then washed with PBST and patted dry;

[0066] (4) Add 100 μL of colorimetric solution to each well, place the plate at 37°C for 15 min, then add 50 μL of 2 M H2SO4 stop solution to each well and measure the absorbance of the solution at 450 nm using an ELISA reader.

[0067] 3. Optimization of the dilution multiple of the platinum-palladium-rhodium probe and the concentration of the coated antigen:

[0068] (1) Add 100 μL of 5×10 8 , 10 8 , 5×107 , 10 7 , 5×10 6 , 10 6 CFU / mL of Vibrio vulnificus, incubate the ELISA plate at 37°C for 2 h, wash the plate with PBST, and pat dry;

[0069] (2) Add 200 μL of 3% (w / v) skim milk powder to each well, place the ELISA plate at 37°C for 1 h, wash the plate with PBST, and pat dry;

[0070] (3) After the PtPdRh@pAb probe prepared under the optimal conditions was diluted 20, 40, 80, 160, and 320 times, 100 μL per well was added to the well plate, the ELISA plate was incubated at 37 °C for 1 h, and then the plate was washed with PBST and patted dry;

[0071] (4) Add 100 μL of colorimetric solution to each well, place the plate at 37°C for 15 min, then add 50 μL of 2 M H2SO4 stop solution to each well and measure the absorbance of the solution at 450 nm using an ELISA reader.

[0072] Figure 4 The results showed that the best reaction system for coupling platinum palladium rhodium nanozyme with the prepared rabbit polyclonal antibody was aqueous solution. The type of blocking agent would not affect the blocking effect, and the ideal blocking effect could be achieved with a blocking time of 1 hour.

[0073] 4. Establishment of the standard curve of ELISA for detecting Vibrio vulnificus based on platinum-palladium-rhodium probe:

[0074] (1) Add 100 μL of 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 CFU / mL of Vibrio vulnificus, incubate at 37°C for 2 h, then wash the plate with PBST and pat dry;

[0075] (2) Each well was blocked with 200 μL of 3% (w / v) skim milk powder at 37°C for 1 h, then the plate was washed with PBST and patted dry;

[0076] (3) After diluting the PtPdRh@pAb probe 80 times, add 100 μL of probe dilution solution to each well, incubate the ELISA plate at 37 °C for 1 h, then wash the plate with PBST and pat dry;

[0077] (4) Add 100 μL of colorimetric solution to each well, place the plate at 37°C for 15 min, then add 50 μL of 2M H2SO4 stop solution to each well, and use an ELISA reader to measure the absorbance of the solution at 450 nm. According to the P / N value, when the catalytic substrate produces an absorbance value of P / N ≥ 2.1 at 450 nm, it is judged as positive; when P / N < 2.1, it is judged as negative. The data of the absorbance value and the number of colonies are plotted using the origin software and fitted to obtain the standard curve of ELISA for detecting Vibrio vulnificus.

[0078] according to Figure 5 The standard curve and the result of chessboard optimization showed that the antigen concentration reached 10 8 CFU / mL, the curve area is flat, the optimal condition with absorbance value near 1 is selected, and the final probe dilution multiple is 1:80. The LOD of traditional ELISA is calculated to be IC10 = 3.53×10 5 CFU / mL, LOD of direct ELISA method based on PtPdR nanozyme for detection of Vibrio vulnificus = IC10 = 2.94 × 10 5 CFU / mL, the direct ELISA method for detecting Vibrio vulnificus of the present invention has a lower detection limit and reduces the secondary antibody incubation process, which requires less time and has lower cost than traditional ELISA.

[0079] 5. Specificity experiment

[0080] (1) Add 100 μL of 10 9 CFU / mL of Vibrio vulnificus, Listeria monocytogenes, Salmonella, Escherichia coli, and Bacillus cereus were incubated at 37°C for 2 h, then the plates were washed with PBST and patted dry;

[0081] (2) Each well was blocked with 200 μL of 3% (w / v) skim milk powder at 37°C for 1 h, then the plate was washed with PBST and patted dry;

[0082] (3) After diluting the PtPdRh@pAb probe 80 times, add 100 μL of probe dilution solution to each well, incubate the ELISA plate at 37 °C for 1 h, then wash the plate with PBST and pat dry;

[0083] (4) Add 100 μL of colorimetric solution to each well, place the plate at 37°C for 15 min, then add 50 μL of 2M H2SO4 stop solution to each well, and measure the absorbance of the solution at 450 nm using an ELISA reader. According to the P / N value, when the catalytic substrate produces an absorbance value of P / N ≥ 2.1 at 450 nm, it is judged as positive; when P / N < 2.1, it is judged as negative.

[0084] according to Figure 6It can be seen that the direct ELISA method for detecting Vibrio vulnificus of the present invention has high specificity and has no cross reaction with Escherichia coli, Salmonella, Listeria monocytogenes, and Bacillus cereus.

[0085] 6. Stability test

[0086] The PtPdRh@pAb probe was stored at 4 °C for 0, 3, and 7 days.

[0087] (1) Add 100 μL of 10 8 CFU / mL of Vibrio vulnificus were incubated at 37°C for 2 h, then the plates were washed with PBST and patted dry;

[0088] (2) Each well was blocked with 200 μL of 3% (w / v) skim milk powder at 37°C for 1 h, then the plate was washed with PBST and patted dry;

[0089] (3) After diluting the PtPdRh@pAb probe prepared above by 80 times, 100 μL of probe dilution solution was added to each well, and the ELISA plate was incubated at 37°C for 1 h, then washed with PBST and patted dry;

[0090] (4) Add 100 μL of colorimetric solution to each well, place the plate at 37°C for 15 min, then add 50 μL of 2M H2SO4 stop solution to each well, and measure the absorbance of the solution at 450 nm using an ELISA reader. According to the P / N value, when the catalytic substrate produces an absorbance value of P / N ≥ 2.1 at 450 nm, it is judged as positive; when P / N < 2.1, it is judged as negative.

[0091] The complex solution comprises sucrose, trehalose, PEG20000, bovine serum albumin, Tween-20 and PBS; the final concentration of the sucrose is 5%, the final concentration of the trehalose is 2%, the final concentration of the PEG20000 is 1%, the final concentration of the bovine serum albumin is 1%, the final concentration of the Tween-20 is 0.25%, and the final concentration of the PBS is 0.01 mol / mL.

Claims

1. An ELISA kit for detecting Vibrio vulnificus, characterized in that: The method comprises a coating antigen, a blocking agent, a Vibrio vulnificus rabbit polyclonal antibody labeled with a platinum-palladium-rhodium nanocomplex, a PBST plate washer, an enzyme-labeled plate, a PBS buffer, a stop solution, and a TMB color developing solution.

2. An ELISA kit for detecting Vibrio vulnificus according to claim 1, characterized in that: The sealing agent is skimmed milk powder.

3. An ELISA kit for detecting Vibrio vulnificus according to claim 1, characterized in that: The Vibrio vulnificus rabbit-derived polyclonal antibody is prepared by the following method: inactivated Vibrio vulnificus is mixed with Freund's adjuvant to prepare a vaccine, the vaccine is used to immunize experimental rabbits at intervals, the serum of the experimental rabbits is collected, and the Vibrio vulnificus polyclonal antibody is extracted and purified from the serum to prepare the Vibrio vulnificus polyclonal antibody.

4. The method for detecting Vibrio vulnificus according to claim 3, characterized in that: The inactivated Vibrio vulnificus is prepared by the following method: resuscitating the Vibrio vulnificus strain and inoculating it into a 3% sodium chloride alkaline water peptone water liquid culture medium for amplification culture, washing the amplified Vibrio vulnificus by centrifugation with PBS, removing the liquid culture medium, and obtaining a Vibrio vulnificus precipitate; finally, resuspending the Vibrio vulnificus precipitate with PBS and adding 1v / v% formaldehyde solution for inactivation culture, washing the Vibrio vulnificus bacterial liquid by centrifugation with PBS, removing the formaldehyde solution, and coating the obtained Vibrio vulnificus bacterial liquid on a nutrient agar plate, and obtaining an inactivated Vibrio vulnificus when no colonies grow.

5. An ELISA kit for detecting Vibrio vulnificus according to claim 1, characterized in that: The method for preparing the Vibrio vulnificus rabbit polyclonal antibody labeled with the platinum-palladium-rhodium nanocomposite is as follows: adding the Vibrio vulnificus rabbit polyclonal antibody to the palladium-rhodium nanocomposite dispersion to react, then adding BSA blocking solution, and finally centrifuging to remove the supernatant to prepare the Vibrio vulnificus rabbit polyclonal antibody labeled with the platinum-palladium-rhodium nanocomposite; The platinum-palladium-rhodium nanocomposite is prepared by the following method: H2PtCl6, Na2PdCl4, RhCl3 and HCl are mixed, poloxamer 407 is added and ultrasonically dissolved, and then L-ascorbic acid is added and reacted at 95°C for 4 hours; after centrifugation, the precipitate is washed with water for multiple times to prepare the platinum-palladium-rhodium nanocomposite.

6. An ELISA kit for detecting Vibrio vulnificus according to claim 5, characterized in that: The method for preparing the Vibrio vulnificus rabbit polyclonal antibody labeled with platinum, palladium, and rhodium nanocomplex is as follows: adding the Vibrio vulnificus rabbit polyclonal antibody to the palladium, and rhodium nanocomplex dispersion liquid to react, then adding BSA blocking liquid, and finally centrifuging to remove the supernatant to prepare the Vibrio vulnificus rabbit polyclonal antibody labeled with platinum, palladium, and rhodium nanocomplex.

7. An ELISA kit for detecting Vibrio vulnificus according to claim 5, characterized in that: The formula of the TMB color developing solution is: 0.5 mL of ethanol solution containing 0.2% w / v 3,3',5,5'-tetramethylbenzidine, 4.5 mL of pure water, 5 mL of phosphate citrate buffer and 10 μL of 30% w / w hydrogen peroxide.

8. An ELISA kit for detecting Vibrio vulnificus according to claim 5, characterized in that: The stop solution is 2M sulfuric acid.

9. A method for detecting Vibrio vulnificus, characterized in that: The detection is performed using the ELISA kit for detecting Vibrio vulnificus described in claim 1.

10. A method for detecting Vibrio vulnificus, characterized in that: The steps are as follows: (1) Add 100 μL of 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 CFU / mL of Vibrio vulnificus, incubate at 37°C for 2 h, then wash the plate with PBST and pat dry; (2) Each well was blocked with 200 μL of 3 w / v% skim milk powder at 37°C for 1 h, then the plate was washed with PBST and patted dry; (3) diluting the Vibrio vulnificus rabbit polyclonal antibody labeled with platinum-palladium-rhodium nanocomplex by 80 times with PBS buffer to obtain a probe diluent, adding 100 μL of the probe diluent to each well of the ELISA plate obtained in step (2), and then incubating the ELISA plate at 37° C. for 1 h, then washing the plate with PBST and patting dry; (4) Add 100 μL of colorimetric solution to each well of the ELISA plate obtained in step (3), place the plate at 37°C for 15 min, then add 50 μL of 2M H2SO4 stop solution to each well, and measure the absorbance of the solution at 450 nm using an ELISA reader. According to the P / N value, when the catalytic substrate produces an absorbance value P / N ≥ 2.1 at 450 nm, it is judged as positive; when P / N < 2.1, it is judged as negative.

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