A nucleic acid aptamer, its derivative and application thereof in detection of lake virus of rohu

Abstract

This invention provides a nucleic acid aptamer, its derivatives, and their application in the detection of tilapia lake virus. The nucleotide sequence of the nucleic acid aptamer is shown in SEQ ID No:1. The derivatives are sequences obtained by substituting, deleting, or adding one or more bases to the nucleotide sequence of the nucleic acid aptamer, or sequences with more than 90% homology to the nucleotide sequence of the nucleic acid aptamer. The nucleic acid aptamer and its derivatives provided by this invention can recognize tilapia lake virus with high affinity and high specificity, enabling rapid, simple, and accurate detection of tilapia lake virus.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and aquatic disease detection technology, specifically relating to a nucleic acid aptamer, its derivatives and their application in detecting tilapia lake virus. Background Technology

[0002] Tilapia is an important aquaculture fish species worldwide, known as the "Fish of the 21st Century" due to its rapid growth, strong adaptability, delicious taste, and high protein content. Currently, tilapia farming is practiced in over 100 countries and regions globally, and the tilapia industry plays a vital role in promoting rural economic development, increasing employment, and generating foreign exchange through exports.

[0003] However, with the continuous expansion and intensification of tilapia farming, disease problems have become increasingly prominent. Tilapia Lake Virus (TiLV) is a newly discovered serious pathogen in recent years, primarily infecting the liver, brain, and other tissues of tilapia, causing symptoms such as lethargy, unsteady swimming, and body ulcers in infected fish, with a mortality rate exceeding 90%. This virus spreads rapidly and has already caused outbreaks in many major tilapia farming countries and regions worldwide, resulting in huge economic losses to the tilapia industry and seriously threatening its sustainable development. Therefore, establishing a rapid, accurate, and convenient method for detecting tilapia lake virus is of great significance for the early diagnosis and precise control of the disease.

[0004] Currently, detection technologies for aquatic animal pathogens mainly include traditional virus isolation and identification, electron microscopy, and molecular biology methods such as nucleic acid detection. Among these, polymerase chain reaction (PCR), nested PCR, and quantitative real-time PCR (qPCR) have become the "gold standard" for laboratory testing due to their high sensitivity and specificity. In addition, loop-mediated isothermal amplification (LAMP) technology, due to its isothermal amplification, rapid reaction, and suitability for on-site diagnosis, has also been reported for the immediate detection of pathogens. However, all of the above methods rely on high-quality nucleic acid extraction as a prerequisite and have limitations such as cumbersome operation procedures, reliance on sophisticated instruments, and high requirements for professional technicians, making it difficult to meet the rapid screening needs of grassroots farms or field environments. Therefore, developing a simpler, faster, more stable, and more economical new detection strategy is of great significance for the on-site prevention and control of tilapia lake virus diseases. Summary of the Invention

[0005] To address the problems and shortcomings of existing technologies, this invention provides a nucleic acid aptamer, its derivatives, and their application in the detection of tilapia lake virus. The nucleic acid aptamer and its derivatives provided by this invention exhibit high affinity and high specificity for tilapia lake virus, enabling rapid, simple, and accurate detection of the virus.

[0006] According to a first aspect of the present invention, a nucleic acid aptamer and its derivatives are provided, wherein the nucleotide sequence of the nucleic acid aptamer is shown in SEQ ID No:1; the derivative is a sequence obtained by substituting, deleting or adding one or more bases into the nucleotide sequence of the nucleic acid aptamer, or is a sequence having more than 90% homology with the nucleotide sequence of the nucleic acid aptamer.

[0007] The nucleic acid aptamer provided by this invention has a specific nucleotide sequence and the ability to specifically bind to tilapia lake virus. It can be used to specifically identify non-tilapia lake viruses and the detection is rapid, simple and accurate.

[0008] Furthermore, the derivatives of the aforementioned nucleic acid aptamers have sequences with more than 90% homology to the nucleotide sequences of the nucleic acid aptamers. Therefore, they can also specifically bind to tilapia lake viruses and specifically identify non-tilapia lake viruses, making detection equally rapid, simple, and accurate.

[0009] The aforementioned nucleic acid aptamers and their derivatives are non-immunogenic; have small molecular weights, facilitating in vitro chemical synthesis; have short preparation cycles; exhibit good reproducibility; allow for easy modification and substitution of different sites of the nucleic acid aptamers with chemical groups; and possess stable sequences, making them easy to transport and preserve.

[0010] Preferably, the nucleic acid aptamer is single-stranded DNA (ssDNA), double-stranded DNA, or chemically modified DNA; the chemical modification includes at least one of phosphorylation, methylation, amination, thiolation, and isotopization.

[0011] Preferably, the secondary structure of the nucleic acid aptamer is as follows: The secondary structure has a ΔG value of -10.65 KJ / mol. The nucleic acid aptamer provided by this invention tends to spontaneously form secondary structures such as hairpins and stem-loops as shown in the figure, and its Gibbs free energy is -10.65 KJ / mol, indicating good stability. The function of the nucleic acid aptamer (specific recognition of tilapia lake virus) largely depends on its specific three-dimensional spatial structure. This negative ΔG value indicates that, under physiological conditions, the nucleic acid aptamer can stably fold into a functional, active conformation, thereby firmly grasping the viral target and providing a structural basis for accurate virus recognition.

[0012] According to a second aspect of the present invention, a conjugate or marker is provided, comprising the aforementioned nucleic acid aptamer and its derivatives, and a functional substance conjugated or labeled therewith; the functional substance comprises at least one of a fluorescent substance, a luminescent material, biotin, an enzyme, a nanomaterial, and an affinity tag; preferably, the fluorescent substance comprises at least one of 6-carboxy-fluorescein (FAM), fluorescein isothiocyanate (FITC), and carboxytetramethylrhodamine (TAMRA).

[0013] According to a third aspect of the present invention, a kit for detecting tilapia lake virus is provided, the kit comprising any of the above-described nucleic acid aptamers and their derivatives, or the above-described conjugates or markers.

[0014] According to a fourth aspect of the present invention, a biosensor or biochip is provided, the biosensor or biochip comprising any of the nucleic acid aptamers and their derivatives described above, or the conjugates or markers described above.

[0015] According to a fifth aspect of the present invention, a method for screening nucleic acid aptamers according to any one of the preceding claims is provided, the method employing Cell-SELEX technology and comprising the following steps: (1) Provide a random single-stranded DNA nucleotide library; (2) Using tilapia lake virus-infected cells as positive screening targets and uninfected normal cells as negative screening targets; incubating the random single-stranded DNA nucleotide library with the positive screening targets, collecting the bound nucleotide sequences, performing PCR amplification, and obtaining enriched libraries. (3) In subsequent screening rounds, the enriched libraries obtained in the first three rounds of screening are incubated with the reverse screening target to collect unbound nucleotide sequences. (4) Incubate the nucleotide sequences collected in step (4) with the positive screening target and collect the bound nucleotide sequences; (5) Using the nucleotide sequences collected in step (5) as templates, perform PCR amplification to prepare the next generation of secondary libraries; (6) Repeat steps (3) to (5) until enriched with nucleotide sequences that specifically recognize tilapia lake virus; (7) Sequencing the final enriched nucleotide library to obtain the aptamer nucleotide sequence as shown in SEQ ID No:1.

[0016] According to a sixth aspect of the present invention, a method for detecting tilapia lake virus is provided, characterized in that the method includes the step of detecting a sample to be tested using any of the above-described nucleic acid aptamers and their derivatives or the above-described conjugates or markers.

[0017] Preferably, the detection method includes at least one of enzyme-linked nucleic acid aptamer adsorption assay, immunochromatographic test strip method, surface plasmon resonance method, and fluorescence polarization method.

[0018] According to a seventh aspect of the present invention, there is provided the use of the nucleic acid aptamer and its derivatives as described in any of the above claims, or the conjugates or markers described above, in the preparation of a product for detecting tilapia lake virus.

[0019] Preferably, the product used for detecting tilapia lake virus includes at least one of the following applications: kit, biosensor, and biochip.

[0020] According to an eighth aspect of the present invention, there is provided the application of the above-described nucleic acid aptamers and their derivatives, or the above-described conjugates or markers, in the detection of tilapia lake virus for non-diagnostic purposes.

[0021] Compared with the prior art, the present invention has the following technical effects: (1) The nucleic acid aptamer (SEQ ID No:1) provided by this invention has extremely high affinity and specificity for tilapia lake virus. Its equilibrium dissociation constant (Kd) is 326.4 nM, and it can accurately identify target viruses. It has no cross-reactivity with other aquatic animal viruses such as grouper iridovirus, and the detection limit can reach 5 × 10⁻⁶. 3 It can infect cells at a rate of 100 cells / mL, with high sensitivity.

[0022] (2) Compared with traditional antibodies, the nucleic acid aptamers of the present invention are chemically synthesized single-stranded DNA, which have the advantages of small batch-to-batch variability, low production cost, and short preparation cycle. They are easy to store and transport, and improve the limitation of harsh antibody storage conditions.

[0023] (3) The detection method based on this nucleic acid aptamer does not require nucleic acid extraction and amplification steps, reducing the dependence on precision instruments and professional personnel, and is suitable for high-throughput screening in laboratories and rapid on-site diagnosis in grassroots farms. At the same time, its sequence is easy to be fluorescently or biotinylated, and can be flexibly adapted to a variety of detection platforms. Attached Figure Description

[0024] Figure 1 This is a predicted secondary structure diagram of the nucleotide sequence SEQ ID No:1 in this invention.

[0025] Figure 2 The results of flow cytometry analysis in Example 2 show the specific recognition of TiLV by SEQ ID No:1 labeled with FAM.

[0026] Figure 3 This is the binding affinity verification result for SEQ ID No:1 in Example 3.

[0027] Figure 4 The detection limit analysis results are for SEQ ID No:1 in Example 4. Detailed Implementation

[0028] To enable those skilled in the art to better understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0029] Example 1: Screening and obtaining tilapia lake virus aptamers (SEQ ID No:1) that specifically recognize tilapia. 1.1 Cells and Viruses Tilapia brain cell line (TiB), Tilapia Lake Virus (TiLV).

[0030] 1.2 Random Single-Stranded DNA Library and Primers The initial random single-stranded DNA (ssDNA) library sequence used in this experiment is as follows: 5'-GTCTGAAGTAGACGCAGGAG-N50-ACGCTTACTCAGGTGTGACT-3'.

[0031] N50 represents 50 random nucleotide sequences, with fixed sequences at both ends of the library for PCR amplification. The primers used in the experiment are as follows: The 5' primer sequence 1 is 5'-GTCTGAAGTAGACGCAGGAG-3'; The 5' primer sequence 2 is 5'-FAM-GTCTGAAGTAGACGCAGGAG-3'; The 3' primer sequence 1 is 5'-GAGACTTCATCTGCGTCCTTCG-3'; The 3' primer sequence 2 is 5'-Biotin-ACGCTTACTCAGGTGTGACT-3'.

[0032] 1.3 Preparation of TiB-infected cells 1×10 6 TiB cells per mL were introduced into cell culture plates and cultured overnight. The medium was then replaced with serum-free M199 medium and TiLV cells with an MOI of 1 were introduced. After culturing for another 48 hours, the cells were collected by centrifugation, resuspended in PBS, and stored for later use.

[0033] 1.4 Cell-SELEX The specific operation method of the Cell-SELEX process is as follows: Step A, Construction and synthesis of the initial random single-stranded DNA library (ssDNA random library): Construct a random oligonucleotide library with the sequence: 5'-GTCTGAAGTAGACGCAGGAG-N50-ACGCTTACTCAGGTGTGACT-3', where N50 is a random sequence; Step B: SELEX screening to obtain aptamers that specifically recognize tilapia lake virus: Step B.1: Dissolve the ssDNA random library in 500 μL PBS buffer, denature at 92 °C for 8 min, and then in an ice bath for 5 min. Step B.2: The pretreated ssDNA randomized library from Step B.1 is incubated with TiLV-TiB cells (tilapia brain cells infected with Tilapia lake virus, denoted as TiLV and Tilapia brain cells as TiB) at 4°C in the dark for 40 min. After centrifugation, the supernatant is removed, the cells are washed three times with PBS, resuspended in PBS, and the supernatant is collected to obtain a nucleic acid library that can specifically recognize TiLV infection; this step is the positive screening step. Step C: Using the above nucleic acid library (the supernatant obtained in step B.2) as a template for PCR amplification, the reaction system is as follows: PCR program: 94℃ 2 min, 94℃ 1 min, 60℃ 30 s, 72℃ 1 min, 20 cycles; 72℃ 5 min; to obtain double-stranded dsDNA; the upstream and downstream primer sequences used for amplification are: The 5' primer sequence 1 is 5'-GTCTGAAGTAGACGCAGGAG-3'; The 5' primer sequence 2 is 5'-FAM-GTCTGAAGTAGACGCAGGAG-3'; The 3' primer sequence 1 is 5'-GAGACTTCATCTGCGTCCTTCG-3'; The 3' primer sequence 2 is 5'-Biotin-ACGCTTACTCAGGTGTGACT-3'; Step D: Take 100 μL of streptavidin-coated magnetic beads and incubate them with the PCR amplification product from step C for 30 min; after magnetic separation, discard the supernatant, add 200 μL of 200 mmol / L NaOH solution, incubate at room temperature for 15 min, collect the supernatant, and adjust the pH to neutral with an equimolar amount of HCl. Step E, starting from the 4th round of screening, adds a reverse screening step before the positive screening step. The reverse screening involves incubating the specific library (supernatant) obtained in step D with normal TiB cells (incubate at 4 ℃ for 1 h) and collecting the supernatant. Step F: Repeat the above steps to obtain a nucleic acid library with higher specificity; Step G: The above nucleic acid library is purified and recovered using a PCR purification kit. The obtained ssDNA is the nucleic acid sequence that highly specifically recognizes TiLV infection.

[0034] 1.5 Determination of Nucleic Acid Aptamer Sequence The supernatant collected in section 1.4 was used to assess the recognition ability of TiLV-TiB cells using flow cytometry until the nucleic acid library showed the strongest recognition ability for TiLV-TiB cells. The DNA sample was purified and subjected to high-throughput sequencing. The resulting ssDNA aptamer, which can be used to detect TiLV-TiB cells in this embodiment, has the following nucleotide sequence: 5'-GACGCTTACTCAGGTGTGACTCGAGACTCGGGCGATCTGCACAATTACTCTCTCGGGCATTCGATTAGCCCGAAGGACGCAGATGAAGTCTC-3'.

[0035] 1.6 Predicting the secondary structure of nucleic acid aptamers using MFOLD The obtained nucleic acid aptamer sequences were used for secondary structure prediction using Mfold, available at: https: / / www.unafold.org / mfold / applications / rna-folding-form.php. The prediction results are as follows: Figure 1 In the secondary structure, ΔG = -10.65 KJ / mol.

[0036] Example 2: Flow cytometry analysis of the specificity of SEQ ID No:1 for identifying tilapia lake virus (1) Test materials Tilapia brain cell line (TiB), Tilapia Lake Virus (TiLV), Grouper iridovirus guangxistrain (SGIV), Largemouth bass virus (LMBV), Infectious subcutaneous and hematopoietic necrosis virus (IHHNV).

[0037] (2) Experimental procedures 1×10 6TiB cells per mL were transferred to 6 cm cell culture plates and cultured overnight at 28°C. Control group 1 (negative control) received supernatant and was inoculated with serum-free M199 medium; control group 2 received supernatant and was inoculated with SGIV diluted in serum-free M199 medium; control group 3 received supernatant and was inoculated with LMBV diluted in serum-free M199 medium; control group 4 was inoculated with IHHNV diluted in serum-free M199 medium; and the experimental group consisted of TiLV diluted in serum-free M199 medium. Cells were cultured for another 48 hours, and then collected for subsequent experiments.

[0038] SEQ ID No:1 labeled with hydroxyfluorescein (FAM) was incubated at 92°C for 5 min, followed by an ice bath for 5 min. The denatured probe was added to the collected cells from each group, incubated at 4°C for 30 min, centrifuged at 3000g for 3 min, washed three times with PBS buffer, resuspended in PBS buffer, and the fluorescence changes in the 495 / 535 channels were detected by flow cytometry.

[0039] (3) Test results Figure 2 Flow cytometry analysis was performed to analyze the specific recognition of TiLV by FAM-labeled SEQ ID No:1. The results showed that the fluorescence values ​​in the experimental group were significantly higher than those in the control groups, while the fluorescence values ​​in control groups 2, 3, and 4 were not significantly different from those in control group 1. This indicates that SEQ ID No:1 can specifically identify TiLV-infected TiB cells.

[0040] Example 3: Flow cytometry analysis of the affinity of SEQ ID No:1 for identifying tilapia lake virus (1) Test materials Tilapia brain cell line (TiB), Tilapia Lake Virus (TiLV).

[0041] (2) Experimental procedures FAM-labeled SEQ ID No:1 was denatured at 92 °C for 5 min, followed by an ice bath for 5 min. The treated SEQ ID No:1 was diluted to 2000, 1000, 500, 250, 125, and 62.5 nM, and incubated with TiLV-TiB cells at 4 °C for 30 min. After incubation, cells were collected by centrifugation, washed three times, and resuspended in PBS buffer. Fluorescence changes in channels 495 / 535 were detected by flow cytometry. Under the same conditions, the binding of FAM-labeled SEQ ID No:1 to normal TiB cells served as a control group.

[0042] The fluorescence intensity of the nucleic acid aptamer binding to target cells at each concentration was corrected by subtracting the average fluorescence intensity binding to control cells. The fitted equation is: Y = B max X / (K d + X) Where Y represents the corrected fluorescence intensity of the binding of the nucleic acid aptamer to the target cell at different concentrations (X), and B max K represents the maximum fluorescence intensity at which the nucleic acid aptamer binds to the target cell. d K represents the dissociation constant of the nucleic acid aptamer. d The values ​​were calculated using GraphPadPrism software. All experiments were repeated three times.

[0043] (4) Test results Figure 4 The binding affinity of SEQ ID No:1 was verified, showing that SEQ ID No:1 exhibits high affinity for the target TiLV-TiB cells. The dissociation constant (Kd) of SEQ ID No:1 was calculated to be 506.9 nM.

[0044] Example 4: Detection limit analysis of SEQ ID No:1 for specific recognition of tilapia lake virus (1) Test materials Tilapia brain cell line (TiB), Tilapia Lake Virus (TiLV).

[0045] (2) Experimental procedures TiB-TiLV cells were serially diluted to 1×10⁻⁶ cells using M199 medium. 5 5×10 4 1×10 4 5×10 3 1×10 3 Cells were incubated at 4 °C for 40 min with well-denatured FAM-labeled SEQ ID No:1 aptamer at a density of 1 / mL. After incubation, cells were collected by centrifugation, washed three times, and resuspended in 300 μL PBS for analysis using flow cytometry. Under the same conditions, the binding of FAM-labeled SEQ ID No:1 to normal TiB cells served as a control group.

[0046] TiB cells were serially diluted to 1×10⁻⁶ cells using M199 medium. 5 5×10 4 1×10 4 5×10 3 1×10 3Total RNA was extracted from cells at concentrations of [number] cells / mL using the TriZol kit. The extracted RNA was reverse transcribed into cDNA using the HiFi Script cDNA synthesis kit. Using cDNA as a template and β-actin as an internal control, the expression level of TiLV was quantified. Each sample was tested in triplicate. Primer sequences are as follows: qTiLV-F: 5′-AGTGTGACAGTCGACGCAAT-3′ qTiLV-R: 5′-TCGACGCAGTTAATCCCAGG-3′ TiB-Actin-F: 5′-CTGTCAGCGATGCCAGGGTA-3′ TiB-Actin-R: 5′-GCGGAATCCACGAAACCACC-3′ (3) Test results Figure 4 To evaluate the detection limit analysis results of SEQ ID No:1, the results showed that even at target cell concentrations as low as 5 × 10⁻⁶, the detection limit was within acceptable limits. 3 At a concentration of [number] cells / mL, SEQ ID No:1 still maintains its specific recognition capability, and its sensitivity is comparable to RT-qPCR. Furthermore, for both detection methods, the fluorescence value increases with increasing cell concentration.

[0047] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention, but such modifications or substitutions are all within the scope of protection of the present invention.

Claims

1. A nucleic acid aptamer and its derivatives, characterized in that: The nucleotide sequence of the nucleic acid aptamer is shown in SEQ ID No:1; The derivative is a sequence obtained by substituting, deleting, or adding one or more bases to the nucleotide sequence of the nucleic acid aptamer, or a sequence that has more than 90% homology with the nucleotide sequence of the nucleic acid aptamer.

2. The nucleic acid aptamer and its derivatives as described in claim 1, characterized in that: The nucleic acid aptamer is single-stranded DNA (ssDNA), double-stranded DNA, or chemically modified DNA; the chemical modification includes at least one of phosphorylation, methylation, amination, thiolation, and isotopization.

3. The nucleic acid aptamer and its derivatives as described in claim 1, characterized in that, The secondary structure of the nucleic acid aptamer is shown below: ； Wherein, the ΔG of the secondary structure is -10.65 KJ / mol.

4. A coupling agent or marker, characterized in that: It includes the nucleic acid aptamers and their derivatives as described in claims 1 to 3, as well as functional substances coupled to or labeled therewith; The functional substance includes at least one of fluorescent substances, luminescent materials, biotin, enzymes, nanomaterials, and affinity tags; preferably, the fluorescent substance includes at least one of 6-carboxy-fluorescein (FAM), fluorescein isothiocyanate (FITC), and carboxytetramethylrhodamine (TAMRA).

5. A kit for detecting tilapia lake virus, characterized in that: The kit contains the nucleic acid aptamer and its derivatives as described in any one of claims 1 to 3, or the conjugate or marker as described in claim 4.

6. A biosensor or biochip, characterized in that, The biosensor or biochip comprises the nucleic acid aptamer and its derivatives as described in any one of claims 1 to 3, or the conjugate or marker as described in claim 4.

7. A method for screening nucleic acid aptamers as described in any one of claims 1 to 3, characterized in that, The method employs Cell-SELEX technology and includes the following steps: (1) Provide a random single-stranded DNA nucleotide library; (2) Using tilapia lake virus-infected cells as positive screening targets and uninfected normal cells as negative screening targets; incubating the random single-stranded DNA nucleotide library with the positive screening targets, collecting the bound nucleotide sequences, performing PCR amplification, and obtaining enriched libraries. (3) In subsequent screening rounds, the enriched libraries obtained in the first three rounds of screening are incubated with the reverse screening target to collect unbound nucleotide sequences. (4) Incubate the nucleotide sequences collected in step (4) with the positive screening target and collect the bound nucleotide sequences; (5) Using the nucleotide sequences collected in step (5) as templates, perform PCR amplification to prepare the next generation of secondary libraries; (6) Repeat steps (3) to (5) until enriched with nucleotide sequences that specifically recognize tilapia lake virus; (7) Sequencing the final enriched nucleotide library to obtain the aptamer nucleotide sequence as shown in SEQ ID No:

1.

8. A method for detecting tilapia lake virus, characterized in that, The method includes the step of detecting the sample to be tested using the nucleic acid aptamer and its derivatives as described in any one of claims 1 to 3, or the conjugate or marker as described in claim 4.

9. The detection method according to claim 8, characterized in that, The detection method includes at least one of enzyme-linked nucleic acid aptamer adsorption assay, immunochromatographic test strip method, surface plasmon resonance method, and fluorescence polarization method.

10. The use of the nucleic acid aptamer and its derivatives as described in any one of claims 1 to 3, or the conjugate or marker as described in claim 4, in the preparation of a product for detecting tilapia lake virus.

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