Ochratoxin b nucleic acid aptamer and its fluorescence in situ imaging application

Abstract

The application provides an ochratoxin B nucleic acid aptamer and a fluorescent in situ imaging application thereof. A specific nucleic acid aptamer with high affinity to ochratoxin B is screened and obtained through SELEX technology, and a fluorescent in situ imaging technology based on primer strand displacement amplification signal amplification is constructed based on the nucleic acid aptamer. The sensor uses the target ochratoxin B nucleic acid aptamer as a recognition element, carries the primer strand displacement amplification technology to amplify the detection signal, so that in-situ detection of ochratoxin B is realized, and the sensor has the advantages of high specificity and simple operation, and provides a new direction for the application of aptamers in fluorescent in situ imaging.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically an ochratoxin B nucleic acid aptamer and its fluorescence in situ imaging application. Background Technology

[0002] Ochratoxin B (OTB) is a common mycotoxin widely found in agricultural products such as grains, animal feed, and wine, as well as their processed foods. Due to its relatively stable chemical properties, it is not easily removed during food processing and storage. Once it enters the body, it may accumulate and cause damage to tissues such as the kidneys, posing certain food safety and health risks.

[0003] Currently, the main methods for detecting ochratoxin B include chromatography, mass spectrometry, immunoassay, and various sensor detection methods. These methods are primarily used for the quantitative analysis of toxin content in samples. While they offer high sensitivity, they typically require complex sample pretreatment or specialized instruments, making it difficult to directly reflect the spatial distribution of ochratoxin B in tissue sections. Furthermore, because ochratoxin B is a small molecule toxin, the preparation and application of specific antibodies are limited, thus restricting its detection by fluorescence in situ imaging.

[0004] Nucleic acid aptamers are single-stranded DNA or RNA obtained through SELEX screening. They can specifically bind to target molecules and have advantages such as ease of synthesis, easy modification, good stability, and suitability for constructing signal amplification systems. Therefore, developing ochratoxin B-specific nucleic acid aptamers and establishing a fluorescence in situ imaging method suitable for tissue sections is of great significance for the localization detection, tissue distribution analysis, and toxicity mechanism research of ochratoxin B. Summary of the Invention

[0005] Given the limitations of existing in-situ imaging detection methods for ochratoxin B in terms of identification elements and detection sensitivity, the technical problem this invention aims to solve is to provide a novel ochratoxin B nucleic acid aptamer and construct a fluorescence in-situ imaging technique based on this aptamer. This sensor has advantages such as high sensitivity, good selectivity, low cost, and ease of operation.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution: On one hand, the present invention provides an ochratoxin B nucleic acid aptamer, which is a single-stranded DNA that specifically recognizes ochratoxin B, selected from any of the following: SEQ ID NO: 1: 5'-AAAATGGAAATCCGAGTGCAAACAGGAACC-3'; SEQ ID NO: 2: 5'-ATCCGAGTGCAAACAGGAA-3'; SEQ ID NO: 3: 5'-CGATGGAAATCCGAAACTATACGAGCTAAA-3'; SEQ ID NO:4: 5'-CGATGGAAATCCGA-3'.

[0007] On the other hand, the present invention provides a fluorescence in situ imaging method for ochratoxin B, comprising the following steps: (1) Preparation of localization probe: The aptamer primer probe shown in SEQ ID NO.9 was mixed with the primer strand displacement amplification system, and amplified at 37℃. After amplification, the polymerase was inactivated by heating to prepare a specific localization probe. (2) Tissue hybridization incubation: Mix the prepared positioning probe with hybridization solution A evenly, drop it onto the surface of the pretreated tissue section, and incubate in the dark; (3) Fluorescent hybridization reaction: The incubated tissue sections were eluted with gradient elution buffer to remove non-specific binding impurities. Then, hybridization solution B containing fluorescently labeled fluorescent probes was added, and the mixture was incubated at 37°C to complete the fluorescent probe hybridization. (4) Staining, mounting and imaging detection: The tissue sections were washed again with elution buffer, stained with nuclear dye, mounted with anti-fluorescence quenching mounting medium, and then observed and acquired with fluorescence in situ imaging signal using a laser confocal scanning microscope to realize the localization detection of ochratoxin B.

[0008] The primer strand displacement amplification system includes: Bst DNA polymerase, dATP, dCTP, dTTP, DNA polymerase buffer, MgSO4, hairpin probe with nucleotide sequence as shown in SEQ ID NO.10, and ochratoxin B aptamer primer probe with nucleotide sequence as shown in SEQ ID NO.9.

[0009] The fluorescent probe is a specific fluorescent probe with the Alexa Fluor 488 fluorescent group labeled at the 5' end and the nucleotide sequence shown in SEQ ID NO.11.

[0010] Hybridization solution A comprises: 10% (w / v) dextran sulfate, 6×SSC buffer, 40% (v / v) deionized formamide, 0.3% lithium dodecyl sulfate, 10% (w / v) Denhardt's solution, and 1% ribonucleoside vanadate complex; hybridization solution B comprises: 10% (w / v) dextran sulfate, 5×SSC buffer, 20% (v / v) deionized formamide, 0.3% lithium dodecyl sulfate, 10% (w / v) Denhardt's solution, and 1% ribonucleoside vanadate complex.

[0011] On the other hand, the present invention provides an ochratoxin B fluorescence in situ imaging detection kit, comprising the aptamer primer probe shown in SEQ ID NO. 9, the hairpin probe shown in SEQ ID NO. 10, the Alexa Fluor 488 labeled fluorescent probe shown in SEQ ID NO. 11, Bst DNA polymerase, dATP, dCTP, dTTP, polymerase buffer, MgSO4, hybridization solution A, hybridization solution B, gradient elution buffer, DAPI staining solution, and anti-fluorescence quenching mounting medium.

[0012] On the other hand, the present invention provides an application of the above-mentioned kit in the detection of ochratoxin B by fluorescence in situ imaging of frozen sections of kidney injury tissue.

[0013] On the other hand, the present invention provides an application of the above-mentioned ochratoxin B nucleic acid aptamer in the detection of ochratoxin B by fluorescence in situ imaging of frozen sections of kidney injury tissue.

[0014] On the other hand, the present invention provides an application of the above-mentioned ochratoxin B nucleic acid aptamer in the histopathological detection of ochratoxin B.

[0015] On the other hand, the present invention provides an application of the above-mentioned ochratoxin B fluorescence in situ imaging method in the histopathological detection of ochratoxin B kidney injury.

[0016] Compared with the prior art, the beneficial effects of the present invention are: 1) This invention is the first to screen and obtain a nucleic acid aptamer sequence with high affinity and specificity for ochratoxin B, providing a novel molecular tool for the identification and detection of ochratoxin B.

[0017] 2) The aptamer sequence is optimized and trimmed to obtain the core binding sequence, which effectively improves the affinity with the target, reduces the sequence length, and is more conducive to probe design and signal amplification.

[0018] 3) Construct a fluorescence in situ imaging technique based on primer strand displacement amplification to amplify the detection signal, significantly improve the detection sensitivity of ochratoxin B, and meet the detection requirements of ultra-low abundance targets in tissue sections.

[0019] 4) Replacing traditional antibodies with nucleic acid aptamers overcomes the technical bottleneck of the lack of available OTB-specific antibodies, while reducing detection costs, shortening detection time, and making the operation simpler.

[0020] 5) Successfully achieved in situ, visualized, and precise localization detection of ochratoxin B in frozen sections of kidney injury tissue, providing a reliable imaging method for the study of in vivo distribution and toxicity of fungal toxins. Attached Figure Description

[0021] Figure 1 Secondary structure diagram of the sequence. a is the secondary structure of the initial sequence 1 of the ochratoxin B aptamer; b is the secondary structure of the trimmed sequence 1 of the ochratoxin B aptamer; c is the secondary structure of the initial sequence 2 of the ochratoxin B aptamer; d is the secondary structure of the trimmed sequence 2 of the ochratoxin B aptamer.

[0022] Figure 2 2D diagram of molecular docking simulation forces. a shows the docking result of ochratoxin B with aptamer initial sequence 1; b shows the docking result of ochratoxin B with aptamer initial sequence 2.

[0023] Figure 3 Colloidal gold colorimetric sensing was used to determine the binding effect of ochratoxin B nucleic acid aptamers before and after cutting.

[0024] Figure 4 Micro-thermal surge technique was used to determine the affinity of ochratoxin B aptamers before and after trimming. a is the initial sequence of ochratoxin B aptamer; b is the trimmed sequence of ochratoxin B aptamer.

[0025] Figure 5 Fluorescence in situ imaging of tissue sections damaged by ochratoxin B. a is a 2D fluorescence in situ imaging image; b is a 3D fluorescence in situ imaging image. Detailed Implementation

[0026] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] Example 1: Screening of ochratoxin B aptamers This invention provides a method for screening ochratoxin B nucleic acid aptamers. This method is based on REase-SELEX exponential enrichment ligand phylogenetic technology and specifically includes the following steps: (1) Initial library construction: A single-stranded DNA initial library containing random sequence regions is synthesized, with fixed primer sequences at both ends, and made complementary to the cDNA library to obtain complementary double-stranded sequences.

[0028] (2) Target incubation and binding: Dissolve the double-stranded sequence in buffer solution, add chlorogenic acid molecules to the system, and incubate at room temperature. The sequence that can be specifically bound by chlorogenic acid will change from the double-stranded complementary state to the single-stranded library-chlorogenic acid complex and the complementary strand.

[0029] (3) Endonuclease and separation: An endonuclease is added to the system after target incubation to cleave the unbound double-stranded complex of chlorogenic acid, which cannot be amplified after cleavage.

[0030] (4) PCR amplification and single-stranded library acquisition: Using the reaction system after endonucleation in step (3) as a template, PCR amplification is performed. The amplification product is recovered, purified and prepared into single-stranded DNA to obtain a secondary library for the next round of screening.

[0031] (5) Repeated screening: The single-stranded DNA product obtained in step (4) is used as the starting library for a new round of screening, and steps (2) to (4) are repeated. After multiple rounds of screening, the library with the highest enrichment is subjected to high-throughput sequencing, and candidate aptamer sequences are obtained through bioinformatics analysis.

[0032] The screening pressure conditions are shown in Table 1.

[0033] Table 1 Screening Pressure Example 2: Optimized trimming and affinity assessment of ochratoxin B aptamers The nucleotide sequences used in the examples are shown in Table 2.

[0034] Table 2 Sequence List The initial sequence 1 (OTB-1) of the ochratoxin B aptamer obtained by screening and sequencing in Example 1, with the sequence: 5'-AAAATGGAAATCCGAGTGCAAACAGGAACC-3' (as shown in SEQ ID NO:1), was subjected to structure prediction and functional domain analysis. The secondary structure prediction diagram is shown below. Figure 1 As shown in a.

[0035] Based on Discovery Studio molecular docking simulation results, it is inferred that the stable binding sites of ochratoxin B molecules may exist at sites A10, T11, C12, A27, and G26. Figure 2 a). Based on this, the ochratoxin B aptamer 1 (OTB1-1) was designed by removing the non-essential end sequences. Its sequence is: 5'-ATCCGAGTGCAAACAGGAA-3' (SEQ ID NO: 2). The secondary structure prediction diagram is shown below. Figure 1 As shown in b.

[0036] The initial sequence 2 (OTB-2) of the ochratoxin B aptamer obtained by screening and sequencing in Example 1, with the sequence: 5'-CGATGGAAATCCGAAACTATACGAGCTAAA-3' (as shown in SEQ ID NO:3), was subjected to structure prediction and functional domain analysis. The secondary structure prediction diagram is shown below. Figure 1 As shown in c.

[0037] Based on the molecular docking simulation results from Discovery Studio, it is inferred that the stable binding sites of ochratoxin B molecules may exist at sites T4, G5, A8, and T10. Figure 2 b). Based on this, the ochratoxin B aptamer 2 (OTB2-1) was designed by removing the non-essential end sequences. Its sequence is: 5'-CGATGGAAATCCGA-3' (SEQ ID NO: 4). The secondary structure prediction diagram is shown below. Figure 1 As shown in d.

[0038] To analyze the properties of ochratoxin B aptamers before and after pruning, a colloidal gold colorimetric sensing platform was established, specifically including the following three aspects: (1) Effect of the target on AuNPs: 10 μL of 100 μM ochratoxin B and 90 μL of AuNPs were added to a 96-well microplate and incubated for 30 min. The same volume of deionized water was used as a control. The absorbance at A520 nm was read using a multi-functional microplate reader. The color change of the system was observed at the same time, and the experiment was repeated 3 times.

[0039] (2) Investigation of NaCl concentration in the reaction system: NaCl solutions of 10, 20, 30, 40, 50, 60 and 70 mM were added to the wells of an enzyme-labeled plate containing 80 μL AuNPs, respectively. The volume was made up to 100 μL with deionized water. After incubation at room temperature for 5 min, the absorption spectrum in the wavelength range of 300-700 nm was scanned with an enzyme-labeled plate reader. The A620 / A520 absorbance ratio was used as the evaluation index. At the same time, the color change of the system was observed. The experiment was repeated 3 times.

[0040] (3) Sequence concentration investigation in the reaction system: Using deionized water instead of the target, the enrichment of AuNPs was investigated at NaCl concentrations of 0, 25, 50, 100, 150 and 200 nM respectively. The sequence concentrations under the above-optimized conditions were added to the wells of the ELISA plate containing 80 μL of AuNPs, and the volume was made up to 100 μL with deionized water. After incubation at room temperature for 15 min, the absorption spectrum in the wavelength range of 300~700 nm was scanned with an ELISA reader. The A620 / A520 absorbance ratio was used as the evaluation index, and the color change of the system was observed at the same time. The experiment was repeated 3 times.

[0041] Analysis of the results from the established colloidal gold colorimetric sensing platform showed that, at ochratoxin B concentrations of 0.1 μM, 0.5 μM, and 2 μM, the affinity of ochratoxin B aptamer 1 (OTB1-1) was increased compared to the uncut version, while the affinity of ochratoxin B aptamer 2 (OTB2-1) was decreased compared to the uncut version. Figure 3 In summary, the ochratoxin B aptamer cut sequence 1 (OTB1-1) exhibits the best binding effect to the target.

[0042] To quantitatively evaluate the trimming effect, affinity was determined using a micro-thermal surge technique. The initial and trimmed sequences were labeled at their 5' ends with Cy5 fluorescent dye, then mixed with a series of ochratoxin B solutions of varying concentrations and detected. By fitting dose-response curves, the Kd value of the initial sequence OTB-1 (SEQ ID NO:1) to ochratoxin B was 17.9 μM, while the Kd value of the optimized trimmed sequence OTB1-1 (SEQ ID NO:2) was 12.0 μM. Figure 4 The affinity was improved to some extent, proving that the tailoring strategy effectively obtained the core aptamer binding sequence with high affinity for ochratoxin B.

[0043] Example 3: Construction of aptamer using in situ fluorescence imaging technique The nucleotide sequences used in the examples are shown in Table 2.

[0044] To verify the feasibility of ochratoxin B aptamers in fluorescence in situ imaging, we first prepared localization probes in test tubes. The reaction system consisted of 800 U / mL Bst DNA polymerase, 600 μM each of dATP, dCTP, and dTTP, 1× polymerase buffer, 10 mM MgSO4, 0.5 μM hairpin probe (SEQ ID NO. 10), and 1 μM ochratoxin B aptamer primer probe (SEQ ID NO. 9). Amplification was performed at 37 ℃ for 1 h. After the reaction, the system was heated at 80 ℃ for 20 min to inactivate Bst DNA polymerase. The localization probe was mixed with hybridization solution A (10% (w / v) dextran sulfate, 6× SSC buffer, 40% (v / v) deionized formamide solution, 0.3% lithium dodecyl sulfate, 10% (w / v) Denhardt's solution, and 1% ribonucleoside vanadate complex) and added dropwise to pretreated frozen tissue sections. The sections were incubated at 40 °C for 12 h. A series of gradient elution buffers were used to elute the tissue sections to reduce background signal and ensure signal accuracy. The slides were placed in hybridization solution B (10% (w / v) dextran sulfate, 5× SSC buffer, 20% (v / v) deionized formamide solution, 0.3% lithium dodecyl sulfate, 10% (w / v) Denhardt's solution, and 1% ribonucleoside vanadate complex), and 5'-terminal labeled Alexa Fluor 488 fluorescent probe (SEQ ID NO. 11) was added to a final concentration of 50 nM. The slides were incubated at 37 °C for 2 h. After incubation, the slides were washed sequentially with elution buffer. Finally, the slides were stained with DAPI for 5 min and mounted with anti-fluorescence quenching mounting medium. The fluorescence signal was observed using a laser confocal scanning microscope. Figure 5 ).

Claims

1. An ochratoxin B nucleic acid aptamer, characterized in that, The nucleic acid aptamer is a single-stranded DNA that specifically recognizes ochratoxin B, and is selected from any of the following: SEQ ID NO: 1: 5'-AAAATGGAAATCCGAGTGCAAACAGGAACC-3'; SEQ ID NO: 2: 5'-ATCCGAGTGCAAACAGGAA-3'; SEQ ID NO: 3: 5'-CGATGGAAATCCGAAACTATACGAGCTAAA-3'; SEQ ID NO:4: 5'-CGATGGAAATCCGA-3'.

2. A method for in situ fluorescence imaging of ochratoxin B, characterized in that, Includes the following steps: (1) Preparation of localization probe: The aptamer primer probe shown in SEQ ID NO.9 was mixed with the primer strand displacement amplification system, and amplified at 37℃. After amplification, the polymerase was inactivated by heating to prepare a specific localization probe. (2) Tissue hybridization incubation: Mix the prepared positioning probe with hybridization solution A evenly, drop it onto the surface of the pretreated tissue section, and incubate in the dark; (3) Fluorescent hybridization reaction: The incubated tissue sections were eluted with gradient elution buffer to remove non-specific binding impurities. Then, hybridization solution B containing fluorescently labeled fluorescent probes was added, and the mixture was incubated at 37°C to complete the fluorescent probe hybridization. (4) Staining, mounting and imaging detection: The tissue sections were washed again with elution buffer, stained with nuclear dye, mounted with anti-fluorescence quenching mounting medium, and then observed and acquired with fluorescence in situ imaging signal using a laser confocal scanning microscope to realize the localization detection of ochratoxin B.

3. The ochratoxin B fluorescence in situ imaging method according to claim 2, characterized in that, The primer chain displacement amplification system comprises: Bst DNA polymerase, dATP, dCTP, dTTP, DNA polymerase buffer, MgSO4, hairpin probe with nucleotide sequence as shown in SEQ ID NO.10, and ochratoxin B aptamer primer probe with nucleotide sequence as shown in SEQ ID NO.

9.

4. The ochratoxin B fluorescence in situ imaging method according to claim 2, characterized in that, The fluorescent probe is a specific fluorescent probe with the Alexa Fluor 488 fluorescent group labeled at the 5' end and the nucleotide sequence shown in SEQ ID NO.

11.

5. The ochratoxin B fluorescence in situ imaging method according to claim 2, characterized in that, Hybridization solution A comprises: 10% (w / v) dextran sulfate, 6×SSC buffer, 40% (v / v) deionized formamide, 0.3% lithium dodecyl sulfate, 10% (w / v) Denhardt's solution, and 1% ribonucleoside vanadium oxide complex; hybridization solution B comprises: 10% (w / v) dextran sulfate, 5×SSC buffer, 20% (v / v) deionized formamide, 0.3% lithium dodecyl sulfate, 10% (w / v) Denhardt's solution, and 1% ribonucleoside vanadium oxide complex.

6. A fluorescence in situ imaging detection kit for ochratoxin B, characterized in that, The kit contains the aptamer primer probe shown in SEQ ID NO. 9, the hairpin probe shown in SEQ ID NO. 10, the Alexa Fluor 488 labeled fluorescent probe shown in SEQ ID NO. 11, Bst DNA polymerase, dATP, dCTP, dTTP, polymerase buffer, MgSO4, hybridization solution A, hybridization solution B, gradient elution buffer, DAPI staining solution, and anti-fluorescence quenching mounting medium.

7. The application of the kit according to claim 6 in the detection of ochratoxin B by fluorescence in situ imaging of frozen sections of kidney injury tissue.

8. The application of the ochratoxin B nucleic acid aptamer according to claim 1 in the fluorescence in situ imaging detection of ochratoxin B in frozen sections of kidney injury tissue.

9. The application of the ochratoxin B nucleic acid aptamer according to claim 1 in the histopathological detection of ochratoxin B.

10. The application of the ochratoxin B fluorescence in situ imaging method according to any one of claims 2-5 in the histopathological examination of kidney injury tissue.

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