Double-enzyme sensing system for detecting ochratoxin A and application of double-enzyme sensing system
By combining amide hydrolase and phenylalanine dehydrogenase, L-phenylalanine is generated and its catalytic product is used for color reaction, which solves the problems of complexity and high cost of existing OTA detection methods and achieves rapid and sensitive detection results.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
- Filing Date
- 2024-11-21
- Publication Date
- 2026-05-22
AI Technical Summary
Existing OTA detection methods suffer from high time costs, complex operation, and the need for large instruments, making it difficult to achieve rapid and sensitive detection.
The combined use of amide hydrolase and phenylalanine dehydrogenase generates L-phenylalanine through hydrolysis, and the color reaction of the phenylalanine dehydrogenase-catalyzed product is used for colorimetric detection. Qualitative and quantitative detection are achieved by combining coenzyme NAD+ and chromogenic reagent MPMS.
It enables efficient, visualized, and quantitative detection of OTA (Over-The-Air) devices, simplifies the operation process, and reduces detection costs.
Smart Images
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of enzyme preparation technology, and relates to a dual-enzyme sensing system for detecting ochratoxin A and its application. Background Technology
[0002] Ochratoxin A (OTA) is a mycotoxin produced by Aspergillus and Penicillium, primarily found in plant-based foods such as grains, dried fruits, and red wine. OTA-contaminated food poses serious health risks to animals and humans through the food chain, exhibiting strong nephrotoxicity, carcinogenicity, and teratogenicity. Due to its high stability, OTA is difficult to remove during food processing. Therefore, developing a rapid and sensitive detection technology is crucial for controlling OTA contamination in food. Current OTA detection methods primarily include liquid chromatography and enzyme-linked immunosorbent assay (ELISA). While these techniques offer high accuracy and sensitivity, they also suffer from drawbacks such as high time costs, the need for large instruments, and complex operation. Recently, biosensors have rapidly emerged as a highly sensitive and convenient detection method. Among various types of biosensors, optical and electrochemical biosensors are the most common.
[0003] Bioenzymes are important macromolecules with multiple catalytic functions, regulating cellular metabolism and maintaining homeostasis in organisms. Their catalytic function and substrate binding are specific, making them a key component of biosensors for sensing or detecting target analytes in complex biological systems. As important elements of optical biosensors, bioenzymes have become a crucial means of detecting target analytes due to their advantages such as simple operation, high sensitivity, and visualization.
[0004] Currently, various functional bioenzymes derived from microorganisms have been found to degrade OTA, such as carboxypeptidase and amidase. Among them, amidase can hydrolyze OTA into OTα and free L-phenylalanine. In addition, phenylalanine dehydrogenase can, under the action of coenzyme NAD+, bind to and catalyze the product L-phenylalanine to generate phenylpyruvate and NADH. Summary of the Invention
[0005] The technical problem solved by this invention is to provide a method for detecting OTA or products in a sample to be tested.
[0006] To address the aforementioned technical problems, the first aspect of the present invention provides a method for detecting OTA in a sample to be tested, comprising the following steps:
[0007] A1) The sample to be tested was hydrolyzed using an amide hydrolase to obtain the first step reaction solution;
[0008] A2) Add phenylalanine dehydrogenase, coenzyme NAD+, and Glycine to the first step reaction solution, and react again to obtain the second step reaction solution; add a colorimetric reagent to the second step reaction solution to carry out a colorimetric reaction and obtain the reaction product; detect OTA in the sample to be tested based on the reaction product;
[0009] The amidase is any one of the following:
[0010] B1) includes the protein shown in the amino acid sequence of SEQ ID NO: 1;
[0011] The protein shown in B2) is a protein that has a homology of SEQ ID NO: 1 in A1) greater than 99%, 95%, 90%, 85%, or 80% and has the same function.
[0012] B3) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein described in B1) or B2);
[0013] The phenylalanine dehydrogenase is any one of the following:
[0014] C1) includes the protein shown in the amino acid sequence of SEQ ID NO: 3;
[0015] The protein shown in C2) is a protein that has a homology of SEQ ID NO: 3 in B1) greater than 99%, 95%, 90%, 85%, or 80% and has the same function.
[0016] C3) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein described in C1) or C2).
[0017] In the method described above,
[0018] In step A1), the pH value of the test sample hydrolyzed by the amide hydrolase is 7.5.
[0019] In the above text, the buffer solution for the hydrolysis reaction is a solution containing 25 mM Tris and 150 mM NaCl.
[0020] In the method described above,
[0021] In step A2), the detection of OTA in the sample based on the reaction products is a qualitative detection.
[0022] The qualitative detection is any of the following:
[0023] A2-1) Detecting OTA in the test sample based on the color change of the reaction product: Compared with the control group, if the color of the reaction product of the test sample changes (significant change, such as the color developer being MPMS, specifically from red to green), then the test sample contains or is a candidate for containing OTA; if the color of the reaction product of the test sample does not change, then the test sample does not contain or is a candidate for not containing OTA.
[0024] A2-2) Use an ELISA reader to detect the absorbance of the reaction product at OD660nm (applicable to samples containing low concentrations of OTA (less than 1 μM OTA)): Compared with the control group, if the absorbance of the reaction product of the test sample is greater than (significantly greater than) the negative control, then the test sample contains or is a candidate for containing OTA; if the absorbance of the reaction product of the test sample is less than or equal to the negative control, then the test sample does not contain or is a candidate for not containing OTA.
[0025] In the method described above,
[0026] In step A2), the detection of OTA in the sample based on the reaction products is a quantitative detection.
[0027] The quantitative detection involves using an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance of the reaction product at OD660nm, and then inputting it into the OTA standard curve to calculate the OTA concentration.
[0028] The OTA standard curve was prepared by replacing the test sample with different concentrations of OTA.
[0029] Furthermore, the OTA standard curve is y = 0.073 + 0.00162x(R). 2 =0.99)
[0030] In the method described above, the colorimetric agent is MPMS.
[0031] In the method described above, the control group is formed by replacing the test sample with acetonitrile.
[0032] The samples to be tested were OTA (soluble in acetonitrile) or came from food containing added OTA or food contaminated with OTA (such as grains, feed, red wine, coffee or juice).
[0033] In the method described above, the hydrolysis reaction in step A1) is carried out at 37°C for 15 minutes.
[0034] In the method described above, the reaction conditions in step A2) are 37°C for 15 min.
[0035] In the method described above, the conditions for the colorimetric reaction in step A2) are 37°C for 0.5 h.
[0036] In a second aspect, the present invention provides a product for detecting OTA, comprising the amide hydrolase, the phenylalanine dehydrogenase, the coenzyme NAD+, the Glycine, and the chromogenic agent described in the first aspect.
[0037] The products mentioned above are biosensors or reagent kits.
[0038] In the products described above, the detection is a qualitative or quantitative detection.
[0039] Thirdly, the present invention provides the use of the amidase, the phenylalanine dehydrogenase, the coenzyme NAD+, the Glycine, and the chromogenic agent described in the first aspect in any of the following:
[0040] C1) Detect OTA;
[0041] C2) Detect whether the sample contains OTA;
[0042] C3) Detect the OTA content in the sample to be tested;
[0043] C4) Preparation of products for detecting OTA;
[0044] C5) Prepare a product to detect whether the sample to be tested contains OTA;
[0045] C6) Prepare a product for detecting the content of OTA in the sample to be tested.
[0046] Fourthly, the present invention provides for the use of the product described in the second aspect in any of the following:
[0047] C1) Detect OTA;
[0048] C2) Detect whether the sample contains OTA;
[0049] C3) Detect the OTA content in the sample to be tested;
[0050] C4) Preparation of products for detecting OTA;
[0051] C5) Prepare a product to detect whether the sample to be tested contains OTA;
[0052] C6) Prepare a product for detecting the content of OTA in the sample to be tested.
[0053] Experiments of this invention demonstrate that by combining the novel OTA-degrading enzyme ThADH of the OTA sensing element with the product L-phenylalanine sensing element phenylalanine dehydrogenase, the invention achieves efficient, visual, and quantitative detection of OTA through the color reaction during the product catalysis process. Attached Figure Description
[0054] Figure 1 This is a schematic diagram of the composition and detection process of OTA's dual-enzyme sensing system.
[0055] Figure 2 The color change of L-Phe is catalyzed by phenylalanine dehydrogenase.
[0056] Figure 3 Establish L-Phe standard curves and curve functions for MPMS colorimetric reagent.
[0057] Figure 4 Sensitivity detection for ThADH-RdPheDH-MPMS.
[0058] Figure 5 This is a specific detection method for ThADH-RdPheDH-MPMS. Detailed Implementation
[0059] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.
[0060] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.
[0061] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.
[0062] The experimental materials and reagents used in the following examples are as follows: Genes and vectors: Escherichia coli expression vector pET32a(+)(69015-3, Novagen, USA) strain; Escherichia coli BL21(DE3) (Beijing TransGen Biotech Co., Ltd.); enzymes and other biochemical reagents; ochratoxin (OTA, CAS No.: 303-47-9); IPTG; Escherichia coli culture medium TB.
[0063] A pH 7.5 solution containing 25 mM Tris and 300 mM NaCl is obtained by dissolving Tris and NaCl in water, wherein the concentration of Tris is 25 mM and the concentration of NaCl is 300 mM.
[0064] Figure 1 This is a schematic diagram of the composition and detection process of OTA's dual-enzyme sensing system.
[0065] Example 1: ThADH amidase-phenylalanine dehydrogenase biosensor ThADH-RdPheDH-MPMS and OTA detection method
[0066] I. Principle of ThADH amidase-phenylalanine dehydrogenase biosensor for OTA detection
[0067] ThADH amide hydrolase can hydrolyze OTA to produce OTα and L-phenylalanine (L-Phe); phenylalanine dehydrogenase uses the product L-Phe as a substrate and, with the participation of the coenzyme NAD+, generates phenylpyruvate and NADH. 1-Methoxy-5-methylphenazine acid methyl ester salt (1-Methoxy PMS, MPMS) acts as a stable electron transfer medium, which can reduce NADH to NAD+. At the same time, MPMS changes from a pink oxidized form to a non-red reduced form. By measuring the ultraviolet absorbance at the corresponding wavelength using an enzyme-linked immunosorbent assay (ELISA) reader, qualitative or quantitative detection of OTA can be achieved.
[0068] II. Components of the ThADH amidase-phenylalanine dehydrogenase biosensor
[0069] 1. Preparation of thADH, an amino acid hydrolase
[0070] The amino acid sequence of the amide hydrolase ThADH is shown in SEQ ID NO:1.
[0071] The amino acid hydrolase ThADH was prepared using prokaryotic expression, as detailed below:
[0072] The recombinant vector pET32a-Tev-ThADH, expressing the thADH gene encoding the amidotropic enzyme, is obtained by replacing the segment between the BamHI and HindIII restriction sites in plasmid pET32a(+) with the TEV-ThADH gene (SEQ ID NO: 2, positions 490-1725). This places the TEV-ThADH gene downstream of and regulated by the T7 promoter and lac operon. The TrxA tag in this vector is co-expressed with TEV-ThADH to obtain the recombinant TrxA-TEV-ThADH protein.
[0073] The nucleotide sequence of the gene encoding the recombinant TrxA-TEV-ThADH protein is SEQ ID NO:2, wherein positions 1-489 of SEQ ID NO:2 are nucleic acids encoded by the TrxA tag, positions 490-510 of SEQ ID NO:2 are nucleic acids encoded by the TEV restriction site, and positions 511-1725 of SEQ ID NO:2 are nucleic acids encoded by the ThADH protein.
[0074] The recombinant vector pET32a-TEV-ThADH expressing the amide hydrolase ThADH was transformed into *Escherichia coli* BL21(DE3) to obtain recombinant *Escherichia coli* BL21(DE3) / pET-32a-TEV-ThADH. The BL21(DE3) / pET-32a-TEV-ThADH strain was inoculated into 100 mL of LB medium and cultured at 37°C with shaking at 220 rpm for 12 h. Then, it was transferred at a 1% ratio to 5 L of TB medium and cultured at 37°C with shaking at 220 rpm for approximately 6 h (OD600≈0.6). Next, 0.1 mM of the inducer IPTG was added, and the culture was induced at 16°C with shaking at 220 rpm for 16 h. The bacterial cells were then collected by centrifugation. The bacterial cells were resuspended in equilibration buffer (25 mM Tris, 500 mM NaCl, 10% glycerol, pH 7.5). The bacterial cells were lysed using a low-temperature, ultra-high pressure, continuous flow cell disruptor (4℃, 1000 bar, disruption for 30 min, JN-30c low-temperature, ultra-high pressure, continuous flow cell disruptor, Guangzhou Juneng Nanobiotechnology Co., Ltd.) to obtain fragmented bacterial cells.
[0075] Centrifuge 15,000g of the broken bacterial cell fragments for 1 hour to remove the precipitate and collect the supernatant.
[0076] To obtain high-purity enzyme protein, the supernatant was eluted using a protein purification instrument and a nickel-ion affinity chromatography column (buffer A: 25mM Tris, 300mM NaCl, 20mM imidazole, 10% glycerol, pH 7.5; buffer B: 25mM Tris, 300mM NaCl, 250mM imidazole, 10% glycerol, pH 7.5, solvent: water). 80% of the eluent from buffer B was collected to obtain the target protein. 200μL of TEV protease (Zeye Biotechnology, ZY130873, 300U) was added to the target protein for enzymatic digestion to remove the TrxA tag on the vector. Simultaneously, the target protein was dialyzed in 5L of dialysis buffer (25mM Tris, 300mM NaCl, 10% glycerol, pH 7.5, solvent: water). After 3 hours, the dialysis buffer was changed, and the protein was dialyzed overnight at 4°C. The target protein, after enzyme digestion, was passed through a nickel column again, and the protein flowing through without the TrxA tag was collected. The target protein after two purifications was dialyzed in buffer (25 mM Tris, 300 mM NaCl, balance water, pH 7.5), concentrated, collected, and stored at -80℃ to obtain the thADH enzyme solution (concentration of 20 mg / mL, solvent of pH 7.5 containing 25 mM Tris and 300 mM NaCl).
[0077] The protein size of the thADH aminohydrolase in the solution was determined to be 45 kDa by SDS-PAGE, consistent with expectations.
[0078] 2. Preparation of phenylalanine dehydrogenase
[0079] The amino acid sequence of phenylalanine dehydrogenase is shown in SEQ ID NO:3, and the nucleotide sequence of the gene it encodes is shown in SEQ ID NO:4.
[0080] Construction of plasmid RdPheDH expressing phenylalanine dehydrogenase: The phenylalanine dehydrogenase encoding gene shown in SEQ ID NO:4 was inserted between the BseRI restriction sites of the pET15-MHL vector (Addgene:26092) to obtain the plasmid.
[0081] The plasmid RdPheDH expressing phenylalanine dehydrogenase was transformed into Escherichia coli BL21(DE3) cells (Full Gold, CD601-03) to obtain the BL21(DE3) / RdPheDH strain. The bacterial culture was evenly spread on a plate with 100 μg / mL ampicillin.
[0082] BL21(DE3) / RdPheDH strain was selected and inoculated into 100 mL of LB medium. The culture was incubated at 37°C and 220 rpm with a shaker until the bacterial OD value reached approximately 0.6. Then, 0.2 mM IPTG was added, and the shaker temperature was lowered to 16°C. Phenylalanine dehydrogenase expression was induced and expressed at 160 rpm for 12 h. The bacterial cells were collected by centrifugation at 5000 rpm for 15 min and resuspended in 25 mM Tris-HCl (pH 7.5) and 500 mM NaCl binding buffer. The cells were then disrupted using an autoclave (4°C, 1000 bar, 30 min) and centrifuged at 12000 rpm for 1.5 h. The supernatant was incubated with Ni-NTA packing material, and phenylalanine dehydrogenase was purified by gradient elution. The target protein was then concentrated and stored at -80°C to obtain a phenylalanine dehydrogenase solution (solvent pH 7.5 containing 25 mM IPTG). A solution of Tris and 300 mM NaCl (concentration 20 mg / mL).
[0083] The protein size of phenylalanine dehydrogenase in the solution was determined to be 38 kDa by SDS-PAGE, consistent with expectations.
[0084] The ThADH amidase-phenylalanine dehydrogenase biosensor includes amidase ThADH and phenylalanine dehydrogenase RdPheDH.
[0085] III. Utilizing phenylalanine dehydrogenase to catalyze L-Phe
[0086] 1. Color change of L-Phe catalyzed by phenylalanine dehydrogenase
[0087] RdPheDH+L-Phe group: The following final concentrations of substances were added to 200 μL of pH 7.5 25 mM Tris-HCl and 150 mM NaCl buffer: 1 mM L-Phe, 100 μg / mL phenylalanine dehydrogenase RdPheDH, 5 mM NAD+ and 5 μM M Lycine, and reacted at 37℃ for 15 min; then, 0.25 mM MPMS was added, and reacted at 37℃ for 30 min; the absorbance at OD 660 nm was measured using a microplate reader, and the absorbance was directly proportional to the L-Phe concentration, thus establishing a standard curve.
[0088] L-Phe group (enzyme-free group): The only difference from the RdPheDH+L-Phe group above is that phenylalanine dehydrogenase RdPheDH is not added.
[0089] Compared with the enzyme-free group, the RdPheDH+L-Phe group changed from red to green if the catalytic reaction proceeded, and showed visible light absorption at an OD wavelength of 660nm.
[0090] The results are as follows Figure 2 As shown, the RdPheDH+L-Phe group exhibits visible light absorption at an OD wavelength of 660nm.
[0091] 2. L-Phe standard curve
[0092] Different final concentrations of L-Phe (0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 μM), 100 μg / mL phenylalanine dehydrogenase RdPheDH, 5 mM NAD+, and 5 μM Glycine were added to 200 μL of 25 mM Tris-HCl and 150 mM NaCl buffer at pH 7.5, respectively, and the mixture was reacted at 37 °C for 15 min. Then, MPMS was added to a final concentration of 0.25 mM and the mixture was reacted at 37 °C for 30 min to obtain the reaction product.
[0093] The color change of the reaction products was observed with the naked eye, and the results were as follows: Figure 3 In the image above, the color changes from red to green as the L-Phe concentration increases.
[0094] The absorbance at OD660nm was measured using an ELISA reader. The absorbance was directly proportional to the L-Phe concentration, and a standard curve was established. The standard curve is shown below. Figure 3 As shown in the image below.
[0095] The higher the L-Phe concentration, the more obvious the color change, and it is directly proportional to the OD660nm absorbance value.
[0096] IV. Construction of a ThADH amidase-phenylalanine dehydrogenase biosensor and establishment of a method for detecting OTA.
[0097] The steps for detecting OTA are as follows:
[0098] 1. Reaction
[0099] Step 1 reaction: OTA degradation by ThADH amide hydrolase: The sample to be tested and ThADH amide hydrolase (prepared in the form of ThADH amide hydrolase solution) were added to a solution containing 25 mM Tris and 150 mM NaCl at pH 7.5. The reaction was carried out in a reactor at 37℃ and 1000 rpm for 15 min to allow OTA to be fully degraded, and the first step reaction solution (containing product L-phenylalanine) was obtained.
[0100] The second step of the reaction was as follows: 100 μg / mL of phenylalanine dehydrogenase (in the form of the phenylalanine dehydrogenase solution prepared in step 2), 5 mM NAD+ (53-84-9 Aladdin), and 5 mM M Glycine (56-40-6 Solarbio) were added to the solution from step 1. The reaction was carried out at 37 °C for 15 min. Then, 0.25 mM MPMS (651652-13-2 Sigma) was added to carry out a colorimetric reaction at 37 °C for 0.5 h to obtain the reaction product.
[0101] 2. Testing
[0102] 1) Qualitative testing
[0103] Visually inspect the reaction product. If the color of the reaction product changes from red before the reaction starts to gray-green, the sample to be tested contains or is a candidate for containing OTA. If the color of the reaction product remains red without change, the sample to be tested does not contain or is a candidate for not containing OTA.
[0104] Alternatively, an ELISA reader can be used to detect the absorbance of the reaction product at OD660nm (this can be used for qualitative analysis of low-concentration OTA). If the absorbance of the reaction product is greater than (significantly greater than) the negative control, the sample contains or is a candidate for containing OTA; if the absorbance of the reaction product is less than or equal to the negative control, the sample does not contain or is a candidate for not containing OTA. The negative control mentioned above involves replacing the sample with acetonitrile.
[0105] 2) Quantitative detection
[0106] The absorbance of the reaction product at OD660nm was detected using an ELISA reader, and then the absorbance was used to calculate the OTA concentration in the OTA standard curve.
[0107] The above OTA standard curve was prepared according to the following method:
[0108] Step 1: Different final concentrations of OTA (0, 30, 60, 90, 120, 150, 180, 210, 240, 270, 300 μM) and ThADH amide hydrolase with a final concentration of 100 μg / mL were added to a solution containing 25 mM Tris and 150 mM NaCl at pH 7.5. The reaction was carried out in a reactor at 37℃ and 1000 rpm for 15 min to allow OTA to be fully degraded, resulting in the first step reaction solution (containing the degradation product L-phenylalanine).
[0109] In the second step, phenylalanine dehydrogenase, NAD+, and Glycine were added to the solution from the first step at a final concentration of 100 μg / mL. The mixture was reacted at 37°C for 15 min, followed by the addition of MPMS at a final concentration of 0.25 mM for a colorimetric reaction at 37°C for 30 min, yielding the reaction product.
[0110] When observed with the naked eye, the color of the reaction product changes with the substrate concentration, gradually turning grayish-green.
[0111] The results are as follows Figure 4 As shown, the method of this invention can detect OTA concentrations in the range of 0-200 μM. Within the range of 0-150 μM, the OTA concentration exhibits a positive linear relationship with the absorbance at an OD wavelength of 660 nm. The OTA standard curve is prepared as y = 0.073 + 0.00162x(R). 2 =0.99), with a detection sensitivity of 150 μMOTA.
[0112] The samples to be tested were OTA (soluble in acetonitrile) or derived from food containing added OTA or food contaminated with OTA (such as grains, feed, red wine, coffee or juice).
[0113] The above-mentioned samples with added OTA were extracted with methanol solution and purified by immunoaffinity chromatography (solvent: 2% acetic acid-98% methanol) to be tested.
[0114] Therefore, the ThADH amidase-phenylalanine dehydrogenase biosensor includes ThADH amidase, phenylalanine dehydrogenase, NAD+, Glycine, MPMS, and a pH 7.5 solution containing 25 mM Tris and 150 mM NaCl.
[0115] V. Specificity of ThADH amidase-phenylalanine dehydrogenase biosensor for detecting OTA toxins
[0116] According to the detection method of number four:
[0117] Step 1: Add different types of mycotoxins (in the form of solutions obtained by dissolving mycotoxins in acetonitrile) to a pH 7.5 solution containing 25 mM Tris and 150 mM NaCl, each with a final concentration of 20 μg / mL. Then add ThADH amide hydrolase with a final concentration of 50 μg / mL. React in a reactor at 37℃ and 1000 rpm / min for 15 min to allow the toxins to be fully degraded, and obtain the reaction solution of step 1.
[0118] The different mycotoxins mentioned above were ochratoxin A (OTA), patulin (PAT), zearalenone (ZEN), deoxynivalenol (DON), or trichothecene toxins (T-2), with acetonitrile used as a negative control.
[0119] The second step of the reaction was to add phenylalanine dehydrogenase (to a final concentration of 100 μg / mL), NAD+ (to a final concentration of 5 mM), and Glycine (to a final concentration of 5 mM) to the solution from the first step of the reaction. The reaction was carried out at 37°C for 15 min. Then, MPMS (651652-13-2Sigma) (to a final concentration of 0.25 mM) was added for a colorimetric reaction at 37°C for 0.5 h to obtain the reaction product.
[0120] The absorbance of the reaction product at OD660nm was measured using an ELISA reader, and the results are as follows: Figure 5 As shown, compared with the negative control group, the absorbance of patulin (PAT), zearalenone (ZEN), deoxynivalenol (DON), and trichothecene toxins (T-2) did not change significantly, while the absorbance of the OTA group was 0.1124, indicating that the method of the present invention specifically detects OTA.
[0121] Substituting the absorbance of the OTA group into the OTA standard curve in section four above, the concentration of OTA was calculated as (0.1124-0.073) / 0.00162 = 24.3 μM. The obtained OTA concentration was consistent with the known concentration, indicating that the method of the present invention can achieve quantitative detection of OTA.
[0122] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.
Claims
1. A method for detecting OTA in a sample, comprising the following steps: A1) The sample to be tested was hydrolyzed using an amide hydrolase to obtain the first step reaction solution; A2) Add phenylalanine dehydrogenase, coenzyme NAD+, and Glycine to the first step reaction solution, and react again to obtain the second step reaction solution; add a colorimetric reagent to the second step reaction solution to carry out a colorimetric reaction and obtain the reaction product; detect OTA in the sample to be tested based on the reaction product; The amidase is any one of the following: B1) includes the protein shown in the amino acid sequence of SEQ ID NO: 1; The protein shown in B2) is a protein that has a homology of SEQ ID NO: 1 in A1) greater than 99%, 95%, 90%, 85%, or 80% and has the same function. B3) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein described in B1) or B2); The phenylalanine dehydrogenase is any one of the following: C1) includes the protein shown in the amino acid sequence of SEQ ID NO: 3; The protein shown in C2) is a protein that has a homology of SEQ ID NO: 3 in B1) greater than 99%, 95%, 90%, 85%, or 80% and has the same function. C3) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein described in C1) or C2).
2. The method according to claim 1, characterized in that: In step A1), the pH value of the test sample hydrolyzed by the amide hydrolase is 7.
5.
3. The method according to claim 1 or 2, characterized in that: In step A2), the detection of OTA in the sample based on the reaction products is a qualitative detection. The qualitative detection is any of the following: A2-1) Detecting OTA in the test sample based on the color change of the reaction product: Compared with the control group, if the color of the reaction product of the test sample changes, then the test sample contains or is a candidate for containing OTA; if the color of the reaction product of the test sample does not change, then the test sample does not contain or is a candidate for not containing OTA. A2-2) Use an ELISA reader to detect the absorbance of the reaction product at OD660nm: Compared with the control group, if the absorbance of the reaction product of the test sample is greater than that of the negative control, then the test sample contains or is a candidate for containing OTA; if the absorbance of the reaction product of the test sample is less than or equal to that of the negative control, then the test sample does not contain or is a candidate for not containing OTA.
4. The method according to claim 1 or 2, characterized in that: In step A2), the detection of OTA in the sample based on the reaction products is a quantitative detection. The quantitative detection involves using an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance of the reaction product at OD660nm, and then inputting it into the OTA standard curve to calculate the OTA concentration. The OTA standard curve was prepared by replacing the test sample with different concentrations of OTA.
5. The method according to any one of claims 1-4, characterized in that: The colorimetric agent is MPMS.
6. A product for detecting OTA, comprising the amide hydrolase of any one of claims 1-5, the phenylalanine dehydrogenase, the coenzyme NAD+, the Glycine, and the chromogenic agent.
7. The product according to claim 6, characterized in that: The product is a biosensor or a reagent kit.
8. The product according to claim 6 or 7, characterized in that: The detection can be qualitative or quantitative.
9. The use of the amidase, the phenylalanine dehydrogenase, the coenzyme NAD+, the Glycine, and the chromogenic agent according to any one of claims 1-5 in any of the following: D1) Detect OTA; D2) Detect whether the sample contains OTA; D3) Detect the OTA content in the sample to be tested; D4) Prepare products for detecting OTA; D5) Prepare a product to detect whether the sample to be tested contains OTA; D6) Prepare a product for detecting the content of OTA in the sample to be tested.
10. The use of the product according to any one of claims 6-8 in any of the following: D1) Detect OTA; D2) Detect whether the sample contains OTA; D3) Detect the OTA content in the sample to be tested; D4) Prepare products for detecting OTA; D5) Prepare a product to detect whether the sample to be tested contains OTA; D6) Prepare a product for detecting the content of OTA in the sample to be tested.