Campylobacter detection method based on glutamyl hydroxamic acid-Fe < 3 + >-ururic acid chromogenic reaction

Abstract

The invention firstly provides an application of glutamyl hydroxamic acid in an iron ion-ururic acid system chromogenic reaction and a glutamyl hydroxamic acid-iron ion-ururic acid chromogenic reaction system. On the basis, the invention provides a colorimetric detection method for campylobacter based on a glutamyl hydroxamic acid-Fe < 3 + >-ururic acid chromogenic reaction. The detection method comprises the following steps: firstly enriching campylobacter in a detection device, then adding a glutamyl hydroxamic acid-Fe < 3 + >-ururic acid chromogenic reaction system, converting cell density into a remarkably amplified color signal, and finally performing color recognition. According to the detection method, by preparing a glutamyl hydroxamic acid-sodium alginate-FsC [Fe < 3 + >] polymer II, amplification conversion from bacterial density to a system color signal is realized, the detection limit is reduced, the sensitivity is improved, the operation is simple, and a feasible scheme for high-throughput detection of campylobacter is provided. The invention further provides a high-throughput campylobacter jejuni detection device, the detection device belongs to a portable biosensing platform, and low-density and high-throughput field detection of campylobacter in shellfish marine products is achieved.

Description

Technical Field

[0001] The present invention belongs to the field of analysis and detection, and relates to a detection method for Campylobacter, and specifically relates to a method based on glutamyl hydroxamic acid-Fe 3+ -Colorimetric detection method for Campylobacter using violuric acid colorimetric reaction. Background Art

[0002] Campylobacter is a heat-resistant pathogenic gram-negative microaerophilic bacterium, including Campylobacter jejuni, Campylobacter gullus, Escherichia coli and Campylobacter Uppsala, which are commonly found in poultry, raw milk and seafood. Among them, Campylobacter jejuni is the representative of Campylobacter and the main pathogen causing human campylobacteriosis; the toxins it produces can cause acute enteritis, such as fever, headache, abdominal pain, diarrhea, bloody stools, nausea, vomiting, etc., and in severe cases, it can also cause Guillain-Barré syndrome. It is worth noting that although the abundance of Campylobacter in food and fecal samples is usually less than 10 3 CFU / g, but only 500 CFU of bacteria can cause symptoms. Therefore, rapid detection of Campylobacter jejuni contamination in food is of great significance for monitoring food safety.

[0003] To solve this problem, researchers conducted research and proposed several solutions. Invention patent application 201911393433.7 discloses "Species-specific molecular targets of Campylobacter jejuni and rapid detection methods thereof". This method uses special primers, PCR amplification and agarose gel electrophoresis detection. If the target band appears in the PCR product, it means that the sample contains Campylobacter jejuni, otherwise it means that the sample does not contain Campylobacter jejuni. Invention patent application 202110802825.5 discloses "A molecular marker and detection method and application of chickens infected with Campylobacter jejuni". By detecting the expression level of isomiR-1, the gga-miR-24-3p isomer of chicken cecal tissue, it is determined whether it is infected with Campylobacter jejuni. Invention patent application 201910841388.0 discloses "A method for detecting Campylobacter jejuni". Based on the magnetic signal generated by the Fe3O4@Au aggregated in the detection area of ​​the test strip, the Campylobacter jejuni content in the sample is quantitatively determined. However, these methods all have problems such as cumbersome steps and long time consumption.

[0004] Seafood is a gift from the sea to mankind and one of the popular diets around the world; however, pathogen contamination has always been a hidden danger to seafood safety and human health. Therefore, rapid detection of Campylobacter jejuni contamination in seafood is an urgent need to test seafood safety and monitor the marine aquaculture environment. Biosensors, especially optical biosensors, have advantages in sensitivity and immediacy, and can achieve accurate, rapid, convenient and cost-effective detection. Among them, colorimetric biosensors have shown great potential in the instant detection of pathogenic bacteria due to their simple operation and naked eye signal readout. However, colorimetric biosensors often have the problem of low sensitivity, which is particularly obvious for low-density analytes. In addition, existing color development systems often require multi-step reactions, time-varying color development, and pre-preparation of new reagents before use, so they cannot meet the needs of low-density and high-throughput bacterial detection.

[0005] Low-density, high-throughput bacterial detection is the most basic requirement for the prevention and control of disease outbreaks, and is crucial to food safety and public health. Currently, there are no reports on the high-throughput, low-density detection of Campylobacter jejuni using a colorimetric system. Summary of the invention

[0006] In view of the current situation that low-density and high-throughput bacterial detection of Campylobacter jejuni in seafood cannot be achieved, the present invention provides a method for detecting Campylobacter jejuni in seafood based on glutamyl hydroxamic acid-Fe 3+ - A colorimetric detection method for Campylobacter using a violuric acid color reaction. The detection method firstly enriches Campylobacter in a detection device, and then adds glutamyl hydroxamic acid-Fe 3+ -violuric acid color development reaction system, converting cell density into significantly amplified color signals, and finally performing color recognition. Compared with the prior art, the detection method not only simplifies the operation steps, but also realizes high-throughput and high-sensitivity on-site detection of Campylobacter, overcomes the shortcomings of the prior art, and has important practical application value.

[0007] The technical solution of the present invention:

[0008] Application of glutamyl hydroxamic acid in the color development reaction of the iron ion-violuric acid system, wherein the maximum absorption peak of the UV-visible spectrum of the complex in the color development reaction is located at 620nm.

[0009] The glutamyl hydroxamic acid-iron ion-violuric acid color reaction system is obtained by sequentially adding and mixing equal volumes of 0.1-1000 μM glutamyl hydroxamic acid solution, iron ion-violuric acid system and MES buffer solution with a pH of 5.5-10.5. The iron ion-violuric acid system is obtained by uniformly mixing 1-20 mM iron ion solution and 25-45 mM violuric acid solution in a volume ratio of 1:3.

[0010] A colorimetric detection and analysis method uses the colorimetric reaction system as described above to perform colorimetric analysis; the specific steps are: combining glutamyl hydroxamic acid with a sample to be tested through reaction, then adding equal volumes of an iron ion-violuric acid system and a MES buffer solution with a pH of 5.5-10.5 to a reaction device in sequence, and finally analyzing the content of the sample to be tested according to color changes.

[0011] A high-throughput, low-density Campylobacter jejuni detection method, which is based on the above-mentioned colorimetric analysis method and specifically comprises the following steps:

[0012] (1) Preparation of a Campylobacter enrichment device: modifying a Campylobacter jejuni specific aptamer onto a 96-well plate. The Campylobacter jejuni specific aptamer is 5'-biotin-Cj1, and the 96-well plate is a transparent and white 96-well plate coated with streptavidin; the specific method is: incubating a 20-100 pmol / mL 5'-biotin-Cj1 solution with a streptavidin-coated 96-well plate for 0.5-3 hours, and discarding the remaining 5'-biotin-Cj1 solution; then incubating a 5-30 mg / mL BSA solution in the 96 wells for 2-12 hours, and discarding the remaining BSA solution, thereby obtaining a Campylobacter enrichment device. The Campylobacter enrichment device can be used to specifically capture and enrich Campylobacter jejuni from actual samples, and the capture efficiency can reach 97.24%, which provides the possibility for low-density detection of Campylobacter.

[0013] (2) Preparation of amplified color development system: First, sodium alginate-FsC[Fe 3+ ]Polymer I, FsC[Fe 3+ ] has a grafting degree of 0.43 nmol / mg; then excess glutamyl hydroxamic acid is dissolved in FsC[Fe 3+ ]-sodium alginate polymer solution. After the reaction is completed, the unreacted glutamyl hydroxamic acid is removed to obtain glutamyl hydroxamic acid-sodium alginate-FsC[Fe 3+ ] polymer II. The polymer II is dissolved in PBS buffer to obtain a polymer II solution with a concentration of 0.5-2.5 mg / mL, i.e., the amplified color system. The FsC[Fe 3+ ] binds to Campylobacter captured in the enrichment device, while the glutamylhydroxamic acid in polymer II converts the bacterial density into an amplified color signal when reacting with the iron ion-violuric acid system.

[0014] Wherein, the glutamyl hydroxamic acid and FsC[Fe 3+The molar ratio of the sodium alginate polymer is 1:1-1:2, thereby ensuring that the amount of grafted glutamyl hydroxamic acid in polymer II is maximized. 3+ ] polymer is specifically as follows: take an appropriate amount of sodium alginate and dissolve it in 0.1M MES buffer (pH=6.0) to obtain a sodium alginate solution system with a concentration of 2 mg / mL; add excess 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and 1-hydroxy-2,5-pyridinedione (NHS) thereto, after the reaction is completed, remove the unreacted EDC and NHS; then, add an appropriate amount of FsC[Fe 3 + ] is added to the sodium alginate solution system; after the reaction is completed, the unreacted FsC[Fe 3+ ], that is, sodium alginate-FsC[Fe 3+ ]polymer.

[0015] (3) Colorimetric detection analysis: First, place the Campylobacter jejuni dispersion to be detected in the Campylobacter enrichment device obtained in step (1), incubate for 20-40 minutes, allow Campylobacter jejuni to bind to the enrichment device, and then pour out the excess dispersion. Then add the amplification color system obtained in step (2), incubate for 10-40 minutes, and pour out the excess amplification color system. Then add equal volumes of iron ion-violuric acid system and MES buffer with pH 5.5-10.5 in sequence, react for 0.5-20 minutes, and measure the absorbance value of the system at a wavelength of 620nm and compare it with the standard curve, or use a color tool for identification, and then obtain the content of Campylobacter. This detection method is not only simple to operate and realizes high-throughput detection, but also has high sensitivity and low detection line, providing a feasible solution for accurately and quickly detecting the content of Campylobacter in seafood.

[0016] The preparation method of the Escherichia jejuni dispersion is as follows: taking an appropriate amount of the sample to be tested, dispersing it in 1×PBS buffer, and stirring it evenly.

[0017] A high-throughput Campylobacter jejuni detection device is composed of a control device and a sample enrichment module I, an amplification and color development module II, and a detection and analysis module III respectively connected to the control device. The sample enrichment device I is a 96-well plate modified with a Campylobacter jejuni specific aptamer. The amplification and color development module II includes a built-in glutamyl hydroxamic acid-sodium alginate-FsC[Fe 3+] device A with a polymer solution, device B with a built-in iron ion-violuric acid system, and device C with a built-in MES buffer of pH 5.5-10.5. The analysis module III is a spectrophotometer or a smartphone camera and color tool program. The high-throughput Campylobacter jejuni detection device described in the present application belongs to a portable biosensor platform that can be used for potential high-throughput and accurate on-site detection of Campylobacter in shellfish marine aquaculture.

[0018] Beneficial effects of the present invention:

[0019] (1) The present invention innovatively proposes a glutamyl hydroxamic acid-iron ion-violuric acid color development reaction system. The color change of the color development reaction system is extremely obvious, and quantitative analysis can be performed by detecting the absorption peak at a wavelength of 620nm or mobile phone color recognition. The operation is simple and has important practical application value.

[0020] (2) Based on the above color development reaction system, the present invention provides a low-density, high-throughput colorimetric detection method for Campylobacter. The detection method comprises preparing glutamyl hydroxamic acid-sodium alginate-FsC[Fe 3+ ]Polymer II realizes the amplified conversion of bacterial density into system color signal, which not only reduces the detection limit and improves the sensitivity, but also is simple to operate, providing a feasible solution for high-throughput detection of Campylobacter.

[0021] (3) Based on the aforementioned detection method, the present application also provides a high-throughput Campylobacter jejuni detection device, which is a portable biosensor platform that realizes low-density, high-throughput on-site detection of Campylobacter in shellfish seafood. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Attached Figure 1 Schematic diagram of the high-throughput Campylobacter jejuni detection device (a) and the schematic diagram of the Campylobacter jejuni detection method (b) described in the present application.

[0023] Attached Figure 2 (a) Photographs and absorbances of glutamyl hydroxamic acid / iron ion-violuric acid reaction products at different concentrations of glutamyl hydroxamic acid in a transparent 96-well plate; (b) Photographs and absorbances of glutamyl hydroxamic acid solutions of different concentrations detected on a transparent 96-well plate using the iodine oxidation method; (cd) Proof of the mechanism of color change caused by the reaction of glutamyl hydroxamic acid with the iron ion-violuric acid complex.

[0024] Attached Figure 3 (ab) GluHx / Fe at different FeCl3 and VA concentrations 3+ -Photographs and absorption spectra of Vio reactants in a transparent 96-well plate; (c) GluHx / Fe 3+-Vio reactants in a transparent 96-well plate after reaction in borax-HCl buffer at different pH values ​​and absorption spectra; (d) GluHx / Fe 3+ -Photographs and absorption spectra of the Vio reactants in a transparent 96-well plate after different reaction times.

[0025] Attached Figure 4 (ab) Cj1 grafting degree of 96-well plate modified with Campylobacter jejuni-specific aptamer (Cj1) at different Cj1 concentrations and reaction times; (cd) nonspecific bacterial binding rate of 96-well plate modified with Campylobacter jejuni-specific aptamer (Cj1) at different BSA concentrations and blocking times.

[0026] Attached Figure 5 Figure 2: (a) Standard curve for qPCR quantification of uncaptured Campylobacter jejuni, where x is the logarithm of the plasmid copy number and y is the corresponding CT value; (b) Cj1-modified 96-well plate for cell density of 1×10 7 CFU / mL enrichment efficiency of Campylobacter jejuni; (c) Cj1 modified 96-well plate for cell density of 1×10 6 Enrichment efficiency of Campylobacter jejuni in CFU / mL.

[0027] Attached Figure 6 (a) In different FsC[Fe 3+ ] concentration, FsC[Fe 3+ ] in GluHx-Alg-FsC[Fe 3+ ]; (b) Grafting rate in different FsC[Fe 3+ ]grafting rate, GluHx / Fe 3+ -Vio reaction solution at 620nm wavelength; (c) Fluorescence intensity of Escherichia coli after incubation with Alg-FsC solutions of different relative molecular weights; (d) 3+ ] / GluHx molar ratio, GluHx in GluHx-Alg-FsC[Fe 3+ ] in the grafting rate.

[0028] Attached Figure 7 (a) Glutamyl hydroxamic acid-sodium alginate-FsC[Fe 3+ ]、FsC[Fe 3+ ], sodium alginate and glutamyl hydroxamic acid; (b) FT-IR spectra of glutamyl hydroxamic acid-sodium alginate-FsC[Fe 3+ ]; (cf) Glutamylhydroxamic acid-sodium alginate-FsC[Fe 3+]’s high-resolution C1s, N 1s, O 1s and Fe 2p spectra.

[0029] Attached Figure 8 (a) Cy5, GluHx, Cy5-modified GluHx (GluHx-Cy5), GluHx-Alg25-FsC[Fe 3+ ] and GluHx-Alg25-FsC[Fe 3+ ]-Cy5 fluorescence spectra; Confocal laser microscopy was used to observe the fluorescence spectra of Campylobacter jejuni with (ce)GluHx-Cy5, (fh)GluHx-Alg25-FsC[Fe 3+ Bright field (BF), fluorescent field (FF), and merged BF-FF images after incubation of ]-Cy5 and (ik)Cy5.

[0030] Attached Fig. 9 GluHx-Alg-FsC[Fe 3+ ] concentration; (b) Designed biosensor GluHx-Alg-FsC[Fe 3+ ] Optimization of incubation time with 96-well plates after capture of Campylobacter jejuni.

[0031] Attached Fig.10 (a) When the cell density is 0, 10, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 CFU / mL, the absorbance spectrum obtained by colorimetric detection of pure culture of Campylobacter jejuni using the designed biosensor; (b) Linear relationship between the cell density of cultured Campylobacter jejuni and the colorimetric intensity of the biosensor; (c) Photo of the single-cell optical tweezers sorting device. (d) Schematic diagram of the single-cell optical tweezers sorting device for sorting single cells of Campylobacter jejuni; (e) The absorbance of Campylobacter jejuni at a cell density of 0-10 CFU / mL was detected using the designed biosensor; (f) The absorbance value of the sensor at a wavelength of 620nm at a cell density of 0-7 CFU / mL; Data are expressed as mean±SD, n=3. a, b, c: Data with different symbols are significantly different.

[0032] Attached Fig.11 (a) Photos of the reaction products of Campylobacter jejuni detected by biosensor at different cell densities, as well as the corresponding RGB images and RGB values ​​read by the smartphone color reading application; (b) 2 , 10 3 , 10 4 , 10 5 and 106 When the CFU / mL was 0.1%, the biosensor was used to detect the change of RGB value of Campylobacter jejuni; the linear relationship between (ce)Log(ΔR / G / B) and Campylobacter jejuni cell density was observed. DETAILED DESCRIPTION

[0033] The present invention will be further described below in conjunction with the embodiments.

[0034] 5'-Biotin-Cj1, synthesized by Sangon Biotech, Shanghai, China. For details, see Liu, W., et al., Talanta, 2024.266.

[0035] Transparent 96-well plates coated with streptavidin were purchased from Thermo Fisher Scientific ( streptavidincoated Clear96-Well Plates, Thermo Scientific, USA).

[0036] Cy5-NHS (Shenzhen Meiluo Technology Co., Ltd., Shenzhen, China)

[0037] Microplate reader (TECAN M200PRO, Switzerland)

[0038] Fluorescence microplate reader (TECAN M200PRO, Switzerland)

[0039] Fourier transform infrared spectroscopy (FT-IR) (PerkinElmer Spectrum 100, PerkinElmer, USA)

[0040] X-ray photoelectron spectroscopy (XPS) (K-Alpha X, Thermo Scientific, US)

[0041] Confocal laser microscope (ZEISS LSM900, Germany)

[0042] Example 1: Study on the mechanism of the reaction between glutamyl hydroxamic acid and iron ion-violuric acid

[0043] (1) Color change phenomenon

[0044] The inventors found in their research that glutamyl hydroxamic acid does not cause any color change when reacting with ferrous ion-violuric acid, but can change the color of ferrous ion-violuric acid. Figure 2As shown in a, when iron ions are mixed with violuric acid (ferric chloride: 10 mM, violuric acid: 40 mM, and the volume ratio of iron chloride to violuric acid is 1:3), the iron ion-violuric acid complex formed exhibits a beautiful light magenta color in aqueous solution, which is different from its dark blue crystalline solid. Therefore, this coloring can provide a clearly recognizable background color without producing heavy color interference. In addition, when the amount of glutamyl hydroxamic acid added exceeds 50 μM, the color of the iron ion-violuric acid solution obviously changes to bright blue, which can be determined by naked eye observation or spectrophotometric measurement ( Figure 2 a). Even at low glutamylhydroxamic acid concentrations (50 μM), this color change can be effectively measured both instrumentally and visually.

[0045] As a control, the inventors used the iodine oxidation method to determine glutamyl hydroxamic acid, and its critical concentration was 500 μM ( Figure 2 b). This indicates that driven by the reaction between glutamyl hydroxamic acid and the iron-violuric acid complex, the method has a fairly high sensitivity for quantifying low concentrations of hydroxamate, thereby achieving a significant reduction in the detection limit and a significant improvement in sensitivity.

[0046] (2) Mechanism studies

[0047] Depend on Figure 2 As can be seen from c and d, the glutamyl hydroxamic acid / ferric ion-violuric acid reactant (glutamyl hydroxamic acid: 1000 μM, ferric ion-violuric acid mixture: a mixture of 10 mM ferric chloride and 40 mM violuric acid in a volume ratio of 1:3) has the same color and spectrophotometric characteristics as the ferrous ion-violuric acid mixed solution (a mixture of 10 mM ferrous chloride and 40 mM violuric acid in a volume ratio of 1:3), and the maximum absorbance peak is located at a wavelength of 620.0 nm. This result shows that the hydroxamic acid group in glutamyl hydroxamic acid has a similar reducing effect to the hydroxylamine group, reducing the iron ions in the ferric ion-violuric acid mixture to generate a dark blue ferrous ion-violuric acid mixture.

[0048] In order to obtain the clearest background color of the glutamyl hydroxamic acid / ferric ion-violuric acid reaction product, the concentration of glutamyl hydroxamic acid was fixed at 1000 μM, and the reaction conditions of the glutamyl hydroxamic acid / ferric ion-violuric acid reaction system were optimized, including the concentrations of ferric chloride and violuric acid, the pH of the borax-hydrochloric acid buffer, and the reaction time. Figure 3 It can be seen that the optimized reaction conditions are 1-20 mM ferric chloride, 25-45 mM violuric acid, pH 5.5-10.5, and reaction time 0.5-20 min.

[0049] Example 2: Preparation of Campylobacter enrichment device and characterization of enrichment performance

[0050] (1) Preparation of Cj1-modified 96-well plate:

[0051] 200 μL of 5′-biotin-Cj1 solution with concentrations of 20, 40, 60, 80, and 100 pmol / mL was incubated with a streptavidin-coated transparent 96-well plate for 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 h, and then the remaining 5′-biotin-Cj1 solution was discarded. 10 μL of 1000×SYBR Green II solution was added to the above-treated 96-well plate, and its fluorescence intensity was measured by a fluorescent microplate reader to determine the grafting degree of the aptamer modified on the well plate. The results showed that the maximum modification amount of the aptamer in a single well was 3.5 pmol ( Figure 4 ab). In addition, according to the above method for preparing a Cj1-modified transparent 96-well plate, a Cj1-modified white 96-well plate was also obtained.

[0052] (2) BSA treatment of Cj1-modified 96-well plate:

[0053] In order to avoid nonspecific binding between free streptavidin in the Cj1-modified 96-well plate and the substance to be detected, bovine serum albumin (BSA) solution was used as a sealant to seal the free streptavidin in the Cj1-modified 96-well plate. The specific operation was as follows: 200 μL of BSA solution with a concentration of 5, 10, 15, 20, 25, and 30 mg / mL was taken in 96 wells and incubated for 2, 4, 6, 8, 10, and 12 hours, and the remaining BSA solution was discarded. The BSA-treated Cj1-modified 96-well plate was incubated with 200 μL of artificial mixed bacterial suspension (including Campylobacter gullus, Campylobacter coli, Helicobacter pylori, Lactobacillus casei, Bifidobacterium, Lactobacillus johnsonii, Enterobacter faecalis, Escherichia coli, Staphylococcus aureus, Streptococcus enteritidis, Lactobacillus monocytogenes, Salmonella gordophylla, Lactobacillus pelvicola, and Streptococcus dysenteriae); the cell density of each bacterium was 1×10 7 CFU / mL, incubate at 37°C for 30 min, and then measure the absorbance of the residual bacterial suspension at 600 nm using a microplate reader.

[0054] like Figure 4 As shown in cd, when the wells were treated with 1.5% (w / v) BSA for 2 hours, the nonspecific binding rate dropped to 3%, while the nonspecific binding rate of the untreated wells was 48%.

[0055] (3) Determination of Campylobacter jejuni enrichment efficiency:

[0056] The cell density was 1×10 7 CFU / mL( Figure 5 b) and 1×10 6 CFU / mL( Figure 5c) 200 μL of Campylobacter jejuni suspension was incubated with the Cj1-modified 96-well plate at 37°C for 5, 10, 15, 20, 25, 30, 35, and 40 min. The remaining Campylobacter jejuni suspension was transferred to an Ep tube, centrifuged at 4000×g for 10 min, and washed three times with 1×PBS buffer (pH=7.2). The number of residual Campylobacter jejuni was determined by fluorescence quantitative PCR. The enrichment efficiency of the Cj1-modified 96-well plate was calculated by formula (1):

[0057]

[0058] Depend on Figure 5 b shows that the cell density is 1×10 7 CFU / mL, the maximum enrichment efficiency is about 50%. 6 CFU / mL, the enrichment efficiency can reach nearly 100% after 20 minutes ( Figure 5 c). It can be seen that in 1×10 1 Up to 1×10 6 In the CFU / mL range, the 96-well plate modified with Cj1 treated with BSA can easily capture more than 97.24% of Campylobacter jejuni cells, and can achieve effective enrichment of Campylobacter in the test system. Based on this, the 96-well plate modified with Cj1 is used as the Campylobacter enrichment device of the present application.

[0059] Example 3: Glutamyl hydroxamic acid-sodium alginate-FsC[Fe 3+ Preparation and characterization of

[0060] (1) Amide bond synthesis of FsC[Fe 3+ ]-Sodium alginate (Mw = 20000-50000) polymer

[0061] Sodium alginate (Alg) was dissolved in 0.1M MES buffer (pH = 6.0) to prepare an Alg solution with a concentration of 2 mg / mL. Subsequently, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and 1-hydroxy-2,5-pyridinedione (NHS) were added to the Alg solution to a final concentration of 2 mg / mL. The above solution was allowed to react for 6 hours. Afterwards, the remaining unreacted EDC and NHS were dialyzed in 1×PBS buffer (pH = 7.2) (molecular weight cutoff: 10000) for 24 hours to remove Alg solution I.

[0062] FsC[Fe 3+ ] was added to the Alg solution I prepared in step (1) to a final concentration of 13.6, 6.8, 3.4, 0.68, and 0.068 μg / mL. The above mixture was reacted for 6 h, and the unreacted FsC[Fe 3+] was dialyzed in 1×PBS buffer (pH=7.2) for 24 h (dialysis bag molecular weight cutoff: 10000) and discarded to obtain sodium alginate-FsC[Fe 3+ ] polymer (hereinafter referred to as "Alg-FsC[Fe 3+ ]”). Finally, the FsC[Fe 3+ ] grafting amount, and calculated FsC[Fe 3 + ]Grafting degree:

[0063]

[0064] According to the results, FsC[Fe 3+ ]grafting degree increases with the FsC[Fe 3+ ] concentration. 3+ ] concentration was 0.68 μg / mL, FsC[Fe 3+ ] The grafting degree is 0.43nmol / mg. Only at this grafting degree can the iron ion-violuric acid color system well indicate 1×10 6 CFU / mL Campylobacter ( Figure 6 ab).

[0065] (2) Glutamyl hydroxamic acid-sodium alginate-FsC[Fe 3+ Preparation of polymer II

[0066] According to FsC[Fe 3+ ]-sodium alginate: glutamyl hydroxamic acid molar ratio is 1:1, 1:1.25, 1:1.5, 1:1.75, 1:2, glutamyl hydroxamic acid is dissolved in FsC[Fe prepared in step (1) 3+ ]FsC[Fe 3 + ]-sodium alginate polymer solution, react for 12 h, then the unreacted glutamyl hydroxamic acid was dialyzed in 1×PBS buffer (pH=7.2) for 24 h and discarded (dialysis bag molecular weight cutoff: 10000) to obtain glutamyl hydroxamic acid-sodium alginate-FsC[Fe 3+ ] polymer II (hereinafter referred to as "GluHx-Alg-FsC[Fe 3+ ]”). The violuric acid-iron ion spectrophotometry was used to determine the activity of the synthesized glutamyl hydroxamic acid-sodium alginate-FsC [Fe 3+ ]The absorbance of the reaction solution at 620 nm.

[0067] According to the results, when GluHx-Alg-FsC[Fe 3+] in Alg-FsC[Fe 3+ ] to GluHx at a molar ratio of 1:1.5, GluHx-Arg-FsC[Fe 3+ ] the GluHx grafting rate reached the maximum value, which can reach 6.94μmol / mg ( Figure 6 d). However, under the experimental conditions of a molar ratio of 1:1-1:2, the number of Campylobacter jejuni in the sample was accurately indicated.

[0068] (3) Effect of the relative molecular mass of sodium alginate on the adhesion ability of non-target bacteria

[0069] The aforementioned GluHx-Alg-FsC[Fe 3+ ] (1.0 mg / mL) and sodium alginate of different molecular weights were dissolved in 1×PBS buffer (pH=7.2); in 1 mL of the system, 1×10 7 The cells were incubated with E. coli at a density of 1000 CFU / mL for 30 minutes at 37°C. The mixtures were then centrifuged at 3000×g for 10 minutes, washed three times with 1× PBS buffer (pH=7.2), and immediately detected by fluorescence microplate reader. ex :450nm,λ em :530nm).

[0070] Figure 6 c shows that due to the high viscosity of sodium alginate solutions with molecular weights of 50,000-100,000 and 100,000-150,000, E. coli and Alg-FsC[Fe 3+ ] after incubation, the fluorescence intensity reached 21.59 and 23.45 au respectively. 3+ ], the optimal molecular weight of sodium alginate is 20,000-50,000; it can ensure the prepared GluHx-Alg-FsC[Fe 3+ ] has an appropriate viscosity to prevent its excessive viscosity from causing nonspecific adhesion to other interfering bacteria, such as Escherichia coli.

[0071] Example 4: Glutamyl hydroxamic acid-sodium alginate-FsC[Fe 3+ ] Characterization and analysis of polymer structure

[0072] Fourier transform infrared spectroscopy (FT-IR, Figure 7 a) and X-ray photoelectron spectroscopy (XPS, Figure 7 bf) Glutamyl hydroxamic acid-sodium alginate-FsC[Fe 3+ ]The chemical structure of polymer II was analyzed.

[0073] Depend on Figure 7 a It can be seen that the infrared spectrum is located at 1647cm -1 、1556cm -1 and 1315cm -1 The characteristic bands at 820 cm-1 correspond to amide I (C=O), amide II (NH), and amide III (CN) groups, respectively, indicating the presence of amide bonds in the product. -1 The peak at 1034cm is the characteristic peak of β-glycosidic bond in sodium alginate. -1 and 1677cm -1 The characteristic peaks of 556 cm-1 correspond to the stretching vibrations of the NO and C=O bonds of the hydroxyl groups in glutamyl hydroxamic acid. -1 The presence of a characteristic peak of iron coordination bond at 3+ ] complexes the iron ions in the resulting polymer. These results indicate that the product is glutamyl hydroxamic acid-sodium alginate-FsC[Fe 3+ ].

[0074] according to Figure 7 b It can be seen that the obtained polymer contains C, O, N and Fe elements. In addition, in the high-resolution C1s spectrum ( Figure 7 c), the peaks at 283.81, 284.69, 286.35, 287.5 and 288.2 eV are C=C, CC, CN, OC=O and NC=O groups respectively. High resolution N1s image ( Figure 7 The peaks at 398.34, 399.77 and 401.45 eV in d) are NO, NC and NH groups respectively. Figure 7 In e), the peaks at 531.29, 532.24, 533.19 and 534.5 eV represent NO, CO, C=O and OH groups, respectively. Figure 7 f) shows the asymmetric bands of 724.25 eV Fe 2p1 / 2 and 710.37 eV Fe 2p3 / 2 generated by spin-orbit splitting, indicating that FsC[Fe 3+ ] in the presence of coordinated iron ions. Overall, the XPS spectra also confirmed the presence of amide bonds, hydroxyl groups, and FsC[Fe 3+ ] group in the obtained polymer, indicating that glutamyl hydroxamic acid-sodium alginate-FsC[Fe 3+ ] were successfully synthesized.

[0075] Example 5: Glutamyl hydroxamic acid-sodium alginate-FsC[Fe 3+ Evaluation of the affinity of polymer II for Campylobacter jejuni

[0076] Using Cyanine5 (Cy5) probe to detect glutamyl hydroxamic acid-sodium alginate-FsC[Fe 3+ ]The affinity of the biopolymers for Campylobacter jejuni was evaluated.

[0077] The specific operation is as follows: 7.82 mg of glutamyl hydroxamic acid-sodium alginate-FsC[Fe 3+ ] and 10.00 mg Cy5-NHS, reacted at 25 ° C for 2 h, and then dialyzed at 4 ° C for 24 h (dialysis bag molecular weight cutoff: 500-1000), and the remaining unreacted Cy5 was discarded to obtain GluHx-Alg-FsC[Fe 3+ ]-Cy5. Subsequently, the resulting solution was freeze-dried and stored at -20°C. Glutamylhydroxamic acid-Cy5 was synthesized as a control.

[0078] The fluorescence microplate reader was used to determine the 3+ ]-Cy5 (10.0 μM), glutamyl hydroxamic acid-Cy5 (10.0 μM) and Cy5 (10.0 μM) solutions at an excitation wavelength (ex) of 646 nm and an emission wavelength (em) of 600-800 nm. 7 CFU / mL of Campylobacter jejuni suspensions were incubated with the above Cy5-labeled polymers (1.0 mg / mL) at 37°C for 30 min. Then, the mixtures were centrifuged at 3000×g for 10 min and washed 3 times with 1×PBS buffer (pH=7.2). Campylobacter jejuni was then resuspended in 1 mL of 1×PBS buffer (pH=7.2) and observed under a confocal laser microscope (maximum wavelength: 646 nm, maximum wavelength: 680 nm).

[0079] Figure 8 ab shows that glutamyl hydroxamic acid-Cy5 and GluHx-Alg-FsC[Fe 3+ ]-Cy5 has the same fluorescence spectrum curve as Cy5, which indicates that Cy5 has been successfully grafted to glutamyl hydroxamic acid and GluHx-Alg-FsC[Fe 3+ ] in polymers. Figure 8 ck showed that, compared with GluHx-Alg-FsC[Fe 3+ jejuni incubated with ]-Cy5 showed red fluorescence, indicating that glutamyl hydroxamic acid-sodium alginate-FsC[Fe 3+ ] biopolymers have a high affinity for Campylobacter jejuni; this is due to the grafting of FsC[Fe 3+Therefore, GluHx-Alg-FsC[Fe 3+ ] can be used as a module of the detection device for affinity to Campylobacter jejuni.

[0080] Example 6: Optimization of colorimetric assay conditions

[0081] (1) Optimization of polymer II solution concentration

[0082] Glutamyl hydroxamic acid-sodium alginate-FsC[Fe 3+ ]Polymer II solution and 1×10 6 CFU / mL concentration of Campylobacter jejuni was incubated in 1 mL of the system for 30 min. Subsequently, the above mixture was centrifuged at 4000×g for 10 min, and the obtained Campylobacter jejuni was washed three times with 1×PBS buffer (pH=7.2). Next, 20 mL of Fe 3+ -Vio reagent and 20mL borax-hydrochloric acid buffer (0.1mol / L, pH=9.50) were added to the Ep tube of Campylobacter jejuni obtained above and reacted for 0.5min. Finally, the absorbance value of the above mixture was measured at 620nm. According to the results, glutamyl hydroxamic acid-sodium alginate-FsC[Fe 3+ The optimal concentration of polymer II was 2 mg / mL ( Fig. 9 a). However, under the experimental conditions of 0.5-2.5 mg / mL, it can accurately indicate the number of Campylobacter jejuni in the sample, meeting the requirements of colorimetric detection and analysis.

[0083] (2) Optimization of incubation time

[0084] In addition, by adding glutamyl hydroxamic acid-sodium alginate-FsC[Fe 3+ ] The incubation time of polymer II with 96-well plate @Cj1-C.jejuni was adjusted to 0, 10, 20, 30, and 40 min. 3+ ] and 96-well plate @Cj1-C.jejuni incubation time of 10-40min can meet the requirements of colorimetric detection analysis ( Fig. 9 b).

[0085] Example 7: High-throughput Campylobacter jejuni detection device

[0086] The high-throughput Campylobacter jejuni detection device is composed of a control device and a sample enrichment module I, an amplification and color development module II, and a detection and analysis module III which are respectively connected to the control device.

[0087] Wherein, the sample enrichment device I is a 96-well plate modified with Campylobacter jejuni specific aptamer. Specific methods and optimization conditions are detailed in Example 2.

[0088] The amplification color development module II includes a built-in glutamyl hydroxamic acid-sodium alginate-FsC[Fe 3+ ] polymer solution, device A with built-in iron ion-violuric acid system, and device C with built-in MES buffer solution of pH 5.5-10.5. 3+ The preparation of the polymer solution is detailed in Example 3, and the optimization of the analysis conditions is detailed in Example 6. The preparation of the iron ion-violuric acid system is detailed in Example 1.

[0089] The analysis module III is a spectrophotometer or a smartphone's camera and color tool program.

[0090] Example 8: Determination of the detection limit of Campylobacter jejuni using the method described in this application

[0091] The cell density was 0 CFU / mL, 1 CFU / mL, 2 CFU / mL, 3 CFU / mL, 4 CFU / mL, 5 CFU / mL, 6 CFU / mL, 7 CFU / mL, 8 CFU / mL, 9 CFU / mL, 1×10 1 CFU / mL, 1×10 2 CFU / mL, 1×10 3 CFU / mL, 1×10 4 CFU / mL, 1×10 5 CFU / mL, 1×10 6 CFU / mL of Campylobacter jejuni suspension were added to transparent 96-well plates @Cj1 and incubated for 20 minutes. Subsequently, the Campylobacter jejuni dispersion that was not bound to the 96-well plate was discarded, and the treated 96-well plate was washed three times with 1× PBS buffer (pH = 7.2). Next, 20 μL of the optimal concentration of GluHx-Alg-FsC[Fe 3+ ] solution and incubated at 37°C for 30 min. Then, the residual GluHx-Alg-FsC[Fe 3+ ] solution, and then 20 μL Fe 3+ -Vio reagent and 20 μL of borax-HCl buffer (0.1 mol / L, pH=9.50), and react for 0.5 min.

[0092] (1) Detection and analysis by measuring absorbance

[0093] The absorbance curve of the mixed solution was measured at 400-800 nm, and a linear regression equation between cell density and the logarithm of absorbance was constructed using Origin 2024b.

[0094] The detection device is 1×10 1 -1×10 6 The response of Campylobacter jejuni in CFU / mL was consistent with the equation [y = 0.2697x-1.9991, R 2 =0.9933]. It is worth noting that the absorption spectra corresponding to these cell densities are clearly discrete at each order of magnitude without any overlap ( Fig.10 ab), which indicates that the detection device has high resolution. More importantly, the obtained equation shows that the theoretical detection limit can be as low as 1 CFU / mL, which is a significant improvement compared with previous work.

[0095] On this basis, this study used a single-cell optical tweezers sorting device to select 1 to 10 Campylobacter jejuni single cells ( Fig.10 cd), and tested the biosensor, and determined that the colorimetric method can clearly identify the signal when the cell density of Campylobacter is 6 CFU / mL and above ( Fig.10 ef), the difference is significant ( Fig.10 f). This shows that the actual detection limit of the developed detection device can be as low as 6 CFU / mL, which is still better than previous studies. This is because the abundant hydroxamic acid groups attached to sodium alginate can show super strong color signals of Campylobacter jejuni cells even at low bacterial density. In addition, the inherent sensitivity of the iron ion-violuric acid reagent to hydroxamic acid also helps to improve the sensitivity of the colorimetric biosensor. This application proposes for the first time a colorimetric detection method that is highly sensitive to Campylobacter jejuni and is independent of bacterial culture and immunoassay, which improves the sensitivity deficiency of the colorimetric biosensor method in the prior art.

[0096] (2) Use smartphone color recognition tools for analysis and testing.

[0097] The transparent 96-well plate was replaced with a white 96-well plate; the smartphone color tool recognition was used for analysis and detection. Specifically, the image was taken by an iPhone 14 Pro with a color reading application embedded in the filter-free mode, and its RGB value was analyzed by the ColorTools (version 1.0) application. The color changes of the system were converted into digital data of R, G and B values ​​( Fig.11 ab). Origin2024b was used to construct a linear regression equation between the logarithm of the cell density and the logarithm of the difference in R, G or B values ​​between each sample and its control.

[0098] Among the three color values, the R value changes most significantly with the difference in cell intensity, indicating that it is the most sensitive response parameter ( Fig.11 ce). The logarithm of ΔR value and the logarithm of cell density are 1×10 1 -1×10 6 There is a linear correlation in the CFU / mL range ( Fig.11 c), which fits the regression equation. Thus, the theoretical detection of the smartphone-assisted method was calculated to be 1 CFU / mL, which also highlights the ultrasensitivity of the biosensor-smartphone platform.

[0099] In addition, this application demonstrates for the first time that the smartphone-assisted detection method of glutamylhydroxamic acid-iron ion-violuric acid system is feasible for sensitive and high-throughput detection of Campylobacter jejuni in 96-well plates.

[0100] Example 9: Biosensor detection of Campylobacter jejuni in spiked Pacific oysters and their aquaculture seawater

[0101] The experiment was repeated three times (n=3) or six times (n=6) as needed, and the results were expressed as mean ± standard deviation. One-way ANOVA was used for mean comparison. P<0.05 was considered statistically significant.

[0102] 1. Detection of Campylobacter jejuni in spiked Pacific oyster (Crassostrea gigas) samples

[0103] The developed smartphone-assisted biosensing platform was used to detect Campylobacter jejuni in spiked Pacific oyster (Crassostrea gigas) samples. First, mucus covering the bodies of these Pacific oysters (uninfected with C. jejuni) was sampled with swabs (10 times), immersed in 1 mL of PBS buffer and stirred for 1 min. Subsequently, Campylobacter jejuni was introduced into the above simulated Pacific oyster samples to a concentration of 1 × 10 1 , 1×10 2 , 1×10 3 , 1×10 4 , 1×10 5 and 1×10 6 In addition, the seawater from which Pacific oysters were cultured was sampled and Campylobacter jejuni was introduced into the cultured seawater sample to a cell density of 1×10 1 , 1×10 2 , 1×10 3 , 1×10 4 , 1×10 5 and 1×10 6 The cell density is expressed as CFU / mL.

[0104] The spiked Pacific oyster and aquaculture seawater samples were added to the transparent and white 96-well plates @Cj1, respectively, and incubated for 20 min. Then, the remaining simulated sample suspension was discarded, and the 96-well plates @Cj1 were washed 3 times with 1× PBS buffer (pH = 7.2). Subsequently, 20 μL of the optimal concentration of GluHx-Alg-FsC[Fe 3+ ] solution and incubated at 37°C for 30 min. Afterwards, the remaining GluHx-Alg-FsC[Fe 3+ ] solution, and then 20 μL Fe 3+ -Vio reagent and 20μL borax-HCl buffer (0.1mol / L, pH=9.50). The above mixture was reacted for 0.5min. Finally, the absorbance value of the samples in the transparent 96-well plate was measured at a wavelength of 620nm. In addition, the samples in the white 96-well plate were photographed using an iPhone 14Pro in filter-free mode, and their RGB values ​​were analyzed using the Color Tools (version 1.0) application. The cell density of Campylobacter jejuni was calculated using Microsoft Excel in the smartphone based on the linear regression equation between the cell density and the logarithm of the difference in absorbance and R value between each sample and its control. At the same time, the number of Campylobacter jejuni in the simulated samples was also determined by qPCR. The detection reproducibility of Campylobacter jejuni was calculated according to formula (3). The test results of the spiked Pacific oyster samples and the aquaculture seawater samples were consistent, and the spiked Pacific oyster samples (see Table 1 for details) are now used as an example for illustration.

[0105] Reproducibility (%) = Smartphone colorimetric measurement result / qPCR measurement result × 100 (3)

[0106] Table 1. Results of smartphone-assisted colorimetric detection of Campylobacter jejuni in spiked Pacific oyster samples

[0107]

[0108] As shown in Table 1, the captured Campylobacter cells in the 96-well plate were detected by smartphone-assisted colorimetry, and the number of Campylobacter jejuni in the mock sample was determined by qPCR. The results were: (1.34±0.42)×10 1 CFU / 100g, (1.74±0.44)×10 2 CFU / 100g, (2.18±0.74)×10 3 CFU / 100g, (2.38±0.72)×10 4 CFU / 100g, (2.40±0.66)×10 5CFU / 100g. The contents determined by qPCR were: (1.96±0.24)×10 1 CFU / 100g, (2.26±0.38)×10 2 CFU / 100g, (2.48±0.70)×10 3 CFU / 100g, (2.62±0.78)×10 4 CFU / 100g, (2.56±0.62)×10 5 CFU / 100g. The recurrence rates were 68.37%, 76.99%, 87.90%, 90.84% ​​and 93.75% respectively.

[0109] This shows that the smartphone-assisted colorimetric detection of Campylobacter cells captured in a 96-well plate has a reproducibility of 68.37-93.75% compared with the qPCR quantitative data, and the higher the Campylobacter content, the higher the reproducibility and the more accurate the test results. This shows that the Campylobacter jejuni detection method described in this application combines smartphone colorimetry with an enrichment device and an amplification color development system to achieve high sensitivity and accuracy in Campylobacter detection in Pacific oysters. In addition, it only takes 15 minutes to complete the processing of these Pacific oyster samples, and only 10 minutes to read the color and output the cell density through a smartphone. The entire detection process only requires simple sample cleaning and the use of a small portable 96-well shaker. It can be seen that the convenience and rapidity of the platform meet the needs of on-site high-throughput detection of Campylobacter in shellfish.

[0110] 2. Detection of Campylobacter jejuni in actual Pacific oysters

[0111] Three batches of Pacific oysters collected from Rushan were taken. The surface sediments were washed with distilled water and the oyster shells were opened. The oyster meat was taken out and weighed. The mucus covering the oyster body was sampled with a swab (10 times), dipped in 1 mL of PBS buffer, stirred for 1 min, and the cell density of Campylobacter jejuni in the PBS buffer was measured using the designed biosensor. The above-treated PBS buffer was added to the transparent and white Cj1-modified 96-well plates, respectively, and incubated for 20 min. The remaining treated PBS buffer was discarded, and the treated Cj1-modified 96-well plates were washed 3 times with 1×PBS buffer (pH=7.2). Subsequently, 20 μL of glutamylhydroxamic acid-sodium alginate-FsC[Fe 3+ ] optimal concentration solution, incubate at 37°C for 30 min, discard the remaining glutamyl hydroxamic acid-sodium alginate-FsC[Fe 3+ ] solution, 20 μL of iron ion-violuric acid reagent and 20 μL of borax-hydrochloric acid buffer (0.1 mol / L, pH=9.50) were added in sequence. The mixture was allowed to react for 0.5 min, and finally the absorption spectrum was obtained.

[0112] In a transparent 96-well plate modified with Cj1, the content of the above mixture was determined at 620 nm using a microplate reader. In addition, the mixed color of the above white 96-well plate @Cj1 was photographed using an iPhone 14 Pro in filter-free mode, and its RGB value was analyzed using the Color Tool (Version 1.0) application. The cell density of Campylobacter jejuni was calculated using the mobile version of Microsoft Excel based on the linear regression equation between the cell density and the logarithm of the difference in absorbance and R value between each sample and the control. At the same time, the number of Campylobacter jejuni in the treated PBS buffer sample was determined by fluorescent quantitative PCR. The method is shown in the supplementary information. Afterwards, the recurrence rate of Campylobacter jejuni detection was calculated according to formula (4). The results are shown in Table 2 for details.

[0113]

[0114] According to the test results in Table 2, fresh Pacific oysters from Rushan, Shandong were selected as samples for the detection of Campylobacter jejuni. The content of Campylobacter in three batches of Pacific oysters was (2.02±0.88)×10 2 CFU / 100g, (1.26±0.66)×10 2 CFU / 100g, (2.78±0.90)×10 1 CFU / 100g. The bacterial concentrations detected by fluorescence quantitative PCR were (3.14±1.08)×10 2 CFU / 100g, (2.44±1.00)×10 2 CFU / 100g、(6.94±1.24)×10 1 CFU / 100g. It can be seen that the recurrence rates of Campylobacter jejuni in Pacific oysters detected by the method described in this application are 64.33%, 51.63% and 40.06%. This shows that the method described in this patent can accurately detect the number of Campylobacter in Pacific oysters.

[0115] Table 2. Results of detecting Campylobacter in Pacific oysters using the method described in this application and fluorescent quantitative PCR

[0116]

[0117] This shows that the glutamyl hydroxamic acid-Fe 3+ The colorimetric detection method of Campylobacter by violuric acid color development reaction provides a practical solution for the rapid, accurate and high-throughput on-site detection of Campylobacter in shellfish. At the same time, the device described in this application also has considerable commercial application prospects.

[0118] In summary, the present invention innovatively proposes a glutamyl hydroxamic acid-iron ion-violuric acid colorimetric reaction system, and based on the aforementioned colorimetric reaction system, provides a low-density, high-throughput colorimetric detection method for Campylobacter. The detection method prepares glutamyl hydroxamic acid-sodium alginate-FsC[Fe 3+ ] Polymer II realizes the amplification conversion of bacterial density to system color signal, which not only reduces the detection limit (6CFU / mL) and improves the sensitivity, but also is simple to operate, greatly shortens the sample detection time, and provides a feasible solution for high-throughput detection of Campylobacter. At the same time, the application also provides a high-throughput Campylobacter jejuni detection device, which belongs to a portable biosensor platform and realizes low-density, high-throughput on-site detection of Campylobacter in shellfish seafood.

Claims

1. Application of glutamyl hydroxamic acid in the color development reaction of iron ion-violuric acid system.

2. The use according to claim 1, characterized in that: The maximum absorption peak of the UV-visible spectrum of the complex in the color development reaction is located at 620nm.

3. Glutamyl hydroxamic acid-iron ion-violuric acid color development reaction system, characterized in that: The color development reaction system is obtained by adding and mixing equal volumes of 0.1-1000 μM glutamyl hydroxamic acid solution, iron ion-violuric acid system and MES buffer solution with a pH of 5.5-10.5 in sequence; the iron ion-violuric acid system is obtained by uniformly mixing 1-20 mM iron ion solution and 25-45 mM violuric acid solution in a volume ratio of 1:

3.

4. A colorimetric analysis method, characterized in that: The colorimetric analysis is performed using the colorimetric reaction system of claim 3, specifically: glutamyl hydroxamic acid is reacted with the sample to be tested, and then an iron ion-violuric acid system and a MES buffer solution with a pH of 5.5-10.5 are added, and the content of the sample to be tested is analyzed according to the color change.

5. A high-throughput, low-density Campylobacter jejuni detection method, characterized in that: The detection method is based on the colorimetric analysis method according to claim 4, and specifically comprises the following steps: (1) Preparation of Campylobacter enrichment device: Modification of Campylobacter jejuni-specific aptamers onto a 96-well plate; (2) Preparation of amplified color development system: First, sodium alginate-FsC[Fe 3+ ] polymer I, where FsC[Fe 3+ ] has a grafting degree of 0.43 nmol / mg; then excess glutamyl hydroxamic acid is dissolved in FsC[Fe 3+ ]-sodium alginate polymer solution; after the reaction is completed, the unreacted glutamyl hydroxamic acid is removed to obtain glutamyl hydroxamic acid-sodium alginate-FsC[Fe 3+ ] polymer II; dissolving polymer II in PBS buffer to obtain a polymer II solution with a concentration of 0.5-2.5 mg / mL, i.e., an amplified color development system; (3) Colorimetric detection analysis: First, place the Campylobacter jejuni dispersion to be detected in the Campylobacter enrichment device obtained in step (1), incubate for 20-40 minutes to allow the Campylobacter jejuni to bind to the enrichment device, and then pour out the excess dispersion; then add the amplification color development system obtained in step (2), incubate for 10-40 minutes, and pour out the excess amplification color development system; then add equal volumes of iron ion-violuric acid system and MES buffer solution with a pH of 5.5-10.5 in sequence, react for 0.5-20 minutes, and measure the absorbance value of the system at a wavelength of 620 nm and compare it with the standard curve, or use a color tool for identification to obtain the Campylobacter content.

6. The method for colorimetric detection of Campylobacter according to claim 5, characterized in that: In step (2), glutamyl hydroxamic acid and FsC[Fe 3+ ]-sodium alginate polymer has a molar ratio of 1:1-1:2; the synthetic sodium alginate-FsC[Fe 3+ ] polymer is specifically as follows: take an appropriate amount of sodium alginate and dissolve it in 0.1M MES buffer (pH=6.0) to obtain a sodium alginate solution system with a concentration of 2 mg / mL; add excess 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and 1-hydroxy-2,5-pyridinedione (NHS) thereto, after the reaction is completed, remove the unreacted EDC and NHS; then, add an appropriate amount of FsC[Fe 3+ ] is added to the sodium alginate solution system; after the reaction is completed, the unreacted FsC[Fe 3+ ], that is, sodium alginate-FsC[Fe 3+ ]polymer.

7. The method for colorimetric detection of Campylobacter according to claim 5, characterized in that: The Campylobacter jejuni-specific aptamer in step (1) is 5'-biotin-Cj1, and the 96-well plate is a transparent 96-well plate coated with streptavidin.

8. The method for colorimetric detection of Campylobacter according to claim 7, characterized in that: The specific method for preparing the Campylobacter enrichment device in step (1) is as follows: incubate 20-100 pmol / mL of 5′-biotin-Cj1 solution with a streptavidin-coated transparent 96-well plate for 0.5-3 h, and discard the remaining 5′-biotin-Cj1 solution; then incubate 5-30 mg / mL of BSA solution in the 96-well plate for 2-12 h, and discard the remaining BSA solution, thereby obtaining a Campylobacter enrichment device.

9. The method for colorimetric detection of Campylobacter according to any one of claims 5 to 8, characterized in that: The preparation method of the Escherichia jejuni dispersion is as follows: taking an appropriate amount of the sample to be tested, dispersing it in 1×PBS buffer, and stirring it evenly.

10. A high-throughput Campylobacter jejuni detection device, characterized in that: The invention is composed of a control device and a sample enrichment module I, an amplification and color development module II, and a detection and analysis module III respectively connected to the control device; the sample enrichment device I is a 96-well plate modified with a Campylobacter jejuni-specific aptamer, the amplification and color development module II includes a built-in glutamyl hydroxamic acid-sodium alginate-FsC[Fe 3+ ] Device A with polymer solution, device B with built-in iron ion-violuric acid system and device C with built-in MES buffer solution of pH 5.5-10.5, and analysis module III is a spectrophotometer or a camera and color tool program for a smartphone.

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