Method for detecting ochratoxin a based on dual-fluorescent dye labeled aptamer

Abstract

The application provides a probe for detecting ochratoxin A, characterized in that the probe comprises an aptamer of ochratoxin A and two fluorescent dye labels, the fluorescent dye labels are located at two ends of the aptamer, and the sequence of the aptamer is GAT CGG GTG TGG GTG GCG TAA AGG GAG CAT CT x wherein X is an integer selected from 0 to 10, and the two fluorescent dye labels can occur fluorescence resonance energy transfer when close in space. The application also provides a detection method of ochratoxin A. The detection method has many advantages, is carried out in a homogeneous solution, does not need separation, does not need a fixed molecule, is fast in detection, stable in signal, simple in operation, high in sensitivity and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological detection, and particularly relates to a method for detecting ochratoxin A based on double-fluorescent dye labeling and a probe used. BACKGROUND

[0002] Ochratoxin A (OTA) is a secondary metabolite of Aspergillus and Penicillium strains. Ochratoxin A can cause pollution of various crops (barley, wheat, corn, peanuts, oats, etc.), nuts, fruits, food, feed and the environment, etc. OTA pollutants are widespread, and related OTA pollution events occur frequently. OTA can enter the human or animal body through the food chain, causing health hazards to humans and animals, and producing various toxicities such as nephrotoxicity, immunotoxicity, teratogenicity and carcinogenicity, etc. Many countries have strict limit standards for the maximum acceptable level of OTA in many foods. Sensitively detecting ochratoxin A is of great significance for food safety, environmental protection, quality control, pollutant screening, human health, etc. The commonly used OTA detection methods often rely on large instruments such as high-performance liquid chromatography, mass spectrometry, etc. Although these instrument methods have advantages such as quantitative accuracy, they also have many shortcomings such as expensive instruments, the need for professional technicians, complex operation steps, time-consuming detection, high detection cost, etc., which are not suitable for rapid on-site detection. Immunoassay technology is also commonly used for OTA detection, which requires the use of antibodies as recognition molecules. The screening and preparation of immune antibodies require animals, etc., and are expensive and have limited stability. Therefore, there is a great demand for low-cost, rapid and sensitive detection of OTA, but also challenges.

[0003] Nucleic acid aptamer as a new type of affinity ligand shows advantages in the field of analysis and sensing. Nucleic acid aptamer can be screened from a random nucleic acid sequence library. Aptamer can be synthesized by chemical method, easy to prepare, good batch reproducibility, and various functional groups (such as fluorescent dyes) can be modified on the aptamer. The detection technology based on nucleic acid aptamer has application prospects in many fields. The use of high-affinity and high-selectivity ochratoxin A aptamer can develop new detection methods for OTA analysis and sensing, such as electrochemical sensing and optical detection. However, many OTA methods based on aptamer still have some limitations, such as the need to fix the aptamer on the electrode surface or the surface of nanomaterials, the need for separation, long detection time, multiple steps, poor signal reproducibility, etc.

[0004] Therefore, there is still a need for improved methods for detecting ochratoxin A. SUMMARY

[0005] The application constructs a double-fluorescent dye-labeled aptamer for analyzing OTA. We label fluorescein (FAM) dye and tetramethyl rhodamine (TMR) dye at two ends of the aptamer sequence that can bind to OTA, as a fluorescent probe for detecting OTA. When detecting OTA, the aptamer fluorescent probe is incubated with OTA, and then the fluorescence intensity of FAM and TMR is measured at the excitation wavelength of FAM. The ratio of the fluorescence intensity of TMR to that of FAM (F TMR / F FAM ) is used as a detection signal for detecting OTA. The fluorescence intensity ratio detection signal (F TMR / F FAM ) of the constructed fluorescently labeled aptamer before and after binding to OTA changes significantly, and OTA can be detected. This ratio-type fluorescence detection mode can overcome the limitation that the signal of the detection method relying solely on the change of fluorescence intensity is prone to fluctuation.

[0006] To achieve the above purpose, the application adopts the following technical solutions:

[0007] The aptamer sequence is reasonably designed, FAM and TMR are labeled at two ends of the aptamer, and the relative distance between FAM and TMR on the aptamer is regulated by changing the length of the aptamer sequence (this relative distance means the degree of spatial closeness, or it can be understood that by inserting several T bases, the ratio of the fluorescence intensity of TMR to that of FAM changes significantly before and after the probe binds to OTA) and the like, so that the ratio of the fluorescence intensity of TMR to that of FAM (F TMR / F FAM ) of the fluorescent dye molecule-labeled aptamer before and after binding to OTA changes significantly (when F TMR / F FAM is used, F TMR / F FAM gradually increases with the increase of the concentration of OTA, and if F FAM / F TMR is used, F FAM / F TMR decreases with the increase of the concentration of OTA), and F TMR / F FAM signal gradually increases with the increase of the concentration of OTA. When detecting OTA, the fluorescent dye-labeled aptamer is incubated with OTA, and then F TMR / F FAM is measured, and the detection of OTA is realized according to the change of F TMR / F FAM signal.

[0008] In some embodiments, the two fluorescent dyes labeled in the application are not limited as long as they can undergo fluorescence resonance energy transfer if they are close in space.

[0009] For the detection of OTA, in some embodiments, the fluorescent dye-labeled aptamer probe is as follows:

[0010] SEQ ID NO: 1 DNA sequence: 5' TMR-GAT CGG GTG TGG GTG GCG TAA AGG GAG CAT C-3' FAM, with tetramethyl rhodamine TMR label at the 5' end and with a fluorescein FAM label at the 3' end, abbreviated as O31-3'FAM-5'TMR.

[0011] SEQ ID NO: 2 DNA sequence: 5' FAM-GAT CGG GTG TGG GTG GCG TAA AGG GAG CAT C-3' TMR, with a FAM label at the 5' end and with a TMR label at the 3' end, abbreviated as O31-5'FAM-3'TMR.

[0012] SEQ ID NO: 3 DNA sequence: 5' TMR-GAT CGG GTG TGG GTG GCG TAA AGG GAG CAT CTTTTT TTT-3' FAM, with a TMR label at the 5' end and with a FAM label at the 3' end, abbreviated as O31-T8-3'FAM-5'TMR.

[0013] SEQ ID NO: 4 DNA sequence: 5' FAM-GAT CGG GTG TGG GTG GCG TAA AGG GAG CAT CTTTTT TTT-3' TMR, with a FAM label at the 5' end and with a TMR label at the 3' end, abbreviated as O31-T8-5'FAM-3'TMR.

[0014] Specifically, the present application provides the following technical solutions:

[0015] In one aspect, the present application provides a probe for detecting ochratoxin A, characterized in that the probe comprises an aptamer of ochratoxin A and two fluorescent dye labels, the fluorescent dye labels are located at both ends of the aptamer, and the two fluorescent dye labels can undergo fluorescence resonance energy transfer when they are close in space.

[0016] In some embodiments, the sequence of the aptamer is GAT CGG GTG TGG GTG GCG TAA AGG GAG CAT CT x , wherein X is an integer selected from 0 to 10.

[0017] In some embodiments, the fluorescent dye labels are FAM and TMR.

[0018] In some embodiments, the 5' end of the aptamer is labeled with FAM and the 3' end is labeled with TMR.

[0019] In some embodiments, X is 8.

[0020] In another aspect, the present application provides a method for detecting ochratoxin A, characterized in that the method comprises mixing the above-mentioned probe with ochratoxin A in a binding buffer solution, incubating at room temperature, and then measuring the fluorescence signal of the sample using a fluorescence spectrometer, and detecting the presence and concentration of ochratoxin A according to the change in the ratio signal of the fluorescence intensity of the two fluorescent dyes.

[0021] In some embodiments, the change in the signal of F 3'末端荧光染料的荧光强度 / F 5'末端荧光染料的荧光强度 is used to detect the presence and concentration of ochratoxin A.

[0022] In some embodiments, the binding buffer comprises 20 mM Tris-HCl (pH 7.5), 120 mM NaCl, and 20 mM CaCl2.

[0023] In another aspect, the present application provides the use of the above-mentioned probe in the preparation of a kit for detecting ochratoxin A.

[0024] In another aspect, the present application provides a kit for detecting ochratoxin A, characterized in that the kit comprises the above-mentioned probe and a binding buffer.

[0025] The present application has the following advantages and effects:

[0026] The present application provides an aptamer labeled with two fluorescent dyes, which can detect ochratoxin A using the ratio signal of the fluorescence intensity of the two fluorescent dyes. The aptamer, which can recognize ochratoxin A with high affinity and high selectivity, is modified with fluorescein (FAM) and tetramethyl rhodamine (TMR) at both ends as a fluorescent probe. Under the same excitation wavelength, the fluorescence intensity of tetramethyl rhodamine and the fluorescence intensity of fluorescein are measured respectively, and the ratio of the fluorescence intensity of tetramethyl rhodamine to the fluorescence intensity of fluorescein is used as the detection signal. The corresponding fluorescence intensity ratio signal changes significantly before and after the probe binds to ochratoxin A, which can realize the detection of the target molecule ochratoxin A. Under optimized experimental conditions, ochratoxin A can be detected with a detection limit of 61 pM. This detection method has many advantages, such as being carried out in a homogeneous solution, not requiring separation or immobilization of molecules, being fast, stable in signal, simple to operate, and highly sensitive. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1The fluorescence signal response of O31-3'FAM-5'TMR probe to OTA is shown, wherein A shows the fluorescence spectrum of blank sample and OTA-containing sample, and B shows the fluorescence intensity ratio signal F TMR / F FAM .

[0028] Figure 2 The F TMR / F FAM signal response of different aptamer fluorescent probes to OTA is compared.

[0029] Figure 3 The results of detecting OTA by using O31-T8-5'FAM-3'TMR are shown.

[0030] Figure 4 The selectivity results of detecting by using O31-T8-5'FAM-3'TMR are shown. DETAILED DESCRIPTION

[0031] In order to make the objectives, technical solutions and advantages of the present application clearer, further detailed description will be made to the present application with reference to the specific embodiments and the accompanying drawings.

[0032] The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The experimental materials and reagents used in the following embodiments are all purchased from conventional reagent companies unless otherwise specified.

[0033] The DNA sequence with fluorescent dye molecule label used is synthesized, prepared and purified by Sheng Wu Bioengineering (Shanghai) Co., Ltd.

[0034] When detecting OTA, the FAM and TMR labeled aptamer probe (final concentration of 50 nM) is mixed with OTA in a binding buffer solution (20 mM Tris-HCl (pH 7.5), 120 mM NaCl, and 20 mM CaCl2), and incubated at room temperature for 30 minutes (or for a shorter time, for example, 10 minutes or less), and then the fluorescence signal of the sample is determined by using a fluorescence spectrometer (JASCO FP-8300, Japan). When scanning the fluorescence spectrum, the excitation wavelength is set to 493 nm, the wavelength scanning range of the fluorescence emission spectrum is 505 nm to 650 nm, the slit width of the excitation light and the emission light is both set to 5 nm, and the fluorescence detection is all carried out at 25℃. The emission fluorescence intensity at 520 nm is determined, which corresponds to the fluorescence intensity F FAM of FAM, and the emission fluorescence intensity at 575 nm is determined, which corresponds to the fluorescence intensity F TMR of TMR. The ratio signal F TMR / FFAM Used for detecting OTA (Over-The-Air) devices.

[0035] Example 1: Changes in SEQ ID NO:1 DNA O31-3'FAM-5'TMR fluorescence signal during OTA binding

[0036] We incubated 50 nM aptamer fluorescent probe O31-3'FAM-5'TMR with OTA (Qingdao Purybang Biotechnology Co., Ltd.) at room temperature for 30 minutes in (20 mM Tris-HCl (pH 7.5), 120 mM NaCl, and 20 mM CaCl2). Then, we used a fluorometer to measure the fluorescence spectra of the blank sample and the sample containing 100 nM OTA, respectively. The results are as follows: Figure 1 As shown in Figure A, the fluorescence spectrum of the blank sample shows fluorescence peaks at 520 nm and 575 nm, corresponding to the fluorescence signals of the fluorescent probe's FAM and TMR, respectively. The fluorescence spectral signals changed significantly before and after the addition of OTA. This is because the aptamer conformation changed after OTA bound to the aptamer fluorescent probe, leading to changes in the fluorescence signal intensities of FAM and TMR. We found that the ratio of fluorescence intensity signal F between the blank sample (the blank sample is the sample without OTA) and the OTA sample was... TMR / F FAM Significantly different (blank sample corresponds to F) TMR / F FAM The value is 1.61, corresponding to the F value for the OTA sample. TMR / F FAM Approximately 1.02), compared to the F of the blank sample. TMR / F FAM In comparison, the F of the OTA sample TMR / F FAM Decrease, F TMR / F FAM The ratio decreased by approximately 0.6. This result indicates that for probe O31-3'FAM-5'TMR, in the presence of OTA, the probe's F... TMR / F FAM reduce.

[0037] Example 2: Fluorescence signal response of different aptamer probes to OTA

[0038] We designed several different probes SEQ ID NO: 1 DNA, SEQ ID NO: 2 DNA, SEQ ID NO: 3 DNA, SEQ ID NO: 4 DNA, we compared the signal response of different fluorescent probes to OTA. 50 nM aptamer fluorescent probe was incubated with OTA in (20 mM Tris-HCl (pH 7.5), 120 mM NaCl, and 20 mM CaCl2) at room temperature for 30 minutes, then the F TMR / F FAM value of blank sample and the F TMR / F FAM value of sample containing 100 nM OTA were measured respectively by fluorescence spectrometer. Figure 2 The F TMR / F FAM signal of different fluorescent probes, blank sample and OTA sample is shown. For blank sample, different fluorescent probes showed different F TMR / F FAM value. In the presence of OTA, different fluorescent probes gave different F TMR / F FAM signal change, among which SEQ ID NO: 1 DNA O31-3'FAM-5'TMR, after the addition of OTA, F TMR / F FAM decreased by about 0.6. SEQ ID NO: 2 DNA O31-5'FAM-3'TMR, after the addition of OTA, F TMR / F FAM decreased by about 0.6. SEQ ID NO: 3 DNA O31-T8-3'FAM-5'TMR, after the addition of OTA, F TMR / F FAM increased slightly, with an increase of about 0.1. SEQ ID NO: 4 DNA O31-T8-5'FAM-3'TMR, before and after the addition of OTA, F TMR / F FAM signal change was the largest, in the presence of OTA, F TMR / F FAM signal increased significantly, with an increase of about 2.6. The corresponding experimental results showed that the designed probe SEQ ID NO: 4 DNA O31-T8-5'FAM-3'TMR had more sensitive signal change, which was the preferred fluorescent probe. It may be that the FAM and TMR labeling positions are more appropriate, and the spatial distance between the two is more appropriate, so the F TMR / F FAM signal increased, and the change amplitude was large, which could be used for the detection of OTA.

[0039] Example 3: Detection of OTA using SEQ ID NO: 4 DNA O31-T8-5'FAM-3'TMR

[0040] SEQ ID NO: 4 DNA O31-T8-5'FAM-3'TMR can be used for the detection of OTA in a ratiometric manner. According to the F TMR / F FAM signal of the probe is changed. We incubated 50 nM O31-T8-5'FAM-3'TMR with OTA in reaction buffer solution (20 mM Tris-HCl (pH 7.5), 120 mM NaCl, and 20 mM CaCl2) at 25 °C for 30 min, and then the F TMR / F FAM signal was measured by a fluorescence spectrometer. The F TMR / F FAM signal gradually increased with the increase of the concentration of OTA, as shown in Fig. 3. The detection range of OTA was 0.061 nM to 500 nM, and the detection limit was 0.061 nM. Figure 3

[0041] Example 4: Investigation of the selectivity of the detection method

[0042] We investigated the selectivity of the detection method for OTA. We investigated a variety of small molecules, including ochratoxin B (OTB), aflatoxin B1 (AFB1), fumonisin B1 (FB1), fumonisin B2 (FB2), and zearalenone (ZAE) (all of the above reagents were purchased from Qingdao Pureban Biological Engineering Co., Ltd.), and detected them under the same experimental conditions. 50 nM SEQ ID NO: 4 DNA O31-T8-5'FAM-3'TMR was incubated with the small molecules under investigation (at a concentration of 200 nM) in reaction buffer solution (20 mM Tris-HCl (pH 7.5), 120 mM NaCl, and 20 mM CaCl2) for 30 min, and then the F TMR / F FAM signal was measured by a fluorescence spectrometer. The F TMR / F FAM signal value (4.42) of the fluorescence probe relative to the blank sample did not change significantly, and was 4.51, 4.43, 4.43, 4.42, and 4.43, respectively, in the presence of other small molecules such as OTB, AFB1, FB1, FB2, and ZAE (at a concentration of 200 nM), while the F TMR / F FAM signal of the fluorescence probe in the presence of 200 nM OTA increased significantly (6.96), indicating that the detection method is selective Figure 4 .

[0043] ​The above-described specific embodiments further illustrate the purpose, technical solutions and beneficial effects of the present application, and it should be understood that the above-described is only a specific embodiment of the present application and is not intended to limit the present application, and any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. A probe for detecting ochratoxin A, characterized in that, The probe comprises an aptamer for ochratoxin A, the aptamer being labeled with fluorescein FAM at its 5' end and tetramethylrhodamine TMR at its 3' end. The sequence of the aptamer is GAT CGG GTG TGG GTG GCG TAA AGG GAG CAT CT x , where X is 8, and the FAM and TMR can undergo fluorescence resonance energy transfer when they are close in space.

2. A kit for detecting ochratoxin A, characterized in that, The kit includes the probe and binding buffer as described in claim 1.

3. The reagent kit according to claim 2, characterized in that, The binding buffer comprises 20 mM Tris-HCl pH 7.5, 120 mM NaCl, and 20 mM CaCl2.

4. A method for detecting ochratoxin A, characterized in that, The method includes mixing the probe of claim 1 with ochratoxin A in a binding buffer solution, incubating at room temperature, and then measuring the fluorescence signal of the sample using a fluorophotometer. The presence and concentration of ochratoxin A are detected based on the change in the ratio signal of the fluorescence intensities of the two fluorescent dyes.

5. The method according to claim 4, characterized in that, Using F TMR / F FAM The presence and concentration of ochratoxin A were detected by detecting changes in the signal.

6. The method according to claim 4 or 5, characterized in that, The binding buffer comprises 20 mM Tris-HCl pH 7.5, 120 mM NaCl, and 20 mM CaCl2.

7. Use of the probe of claim 1 in the preparation of a kit for detecting ochratoxin A.

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