Osteoarthritis early diagnosis kit and construction method thereof

Abstract

The invention discloses an osteoarthritis early diagnosis kit and a construction method thereof, the osteoarthritis early diagnosis kit comprises an osteoarthritis early biomarker cartilage acidic protein targeting aptamer, a capture probe and a recognition probe, and the nucleotide sequence of the aptamer is as shown in SEQ ID NO.1; the capture probe is a reverse complementary strand of a biotin modified aptamer, and the nucleotide sequence of the capture probe is shown as SEQ ID NO. 2; the recognition probe is an aptamer modified by biotin. The method comprises the following steps: sequentially reacting horseradish peroxidase labeled aptamers with a detected sample, fixing complementary chains of the aptamers on a microwell plate, and rapidly determining the content of CRTAC1 based on the color change of an enzyme catalysis substrate. The sequential reaction avoids the excessive combination of the aptamer and the cDNA, so that the full-coverage recognition of the CRTAC1 is ensured; the method shows relatively high sensitivity and stability in determination of CRTAC1 in blood plasma, and has a good standard addition recovery rate.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to an osteoarthritis early diagnosis kit and a construction method thereof. Background Art

[0002] Aptamers are ideal tools for detecting early biomarkers of osteoarthritis. Since serum biomarkers have high sensitivity and specificity and are easy to implement in clinical practice, they have gradually become a widely used means of disease diagnosis. Studies have shown that cartilage acidic protein 1 (CRTAC1) is a new potential biomarker for osteoarthritis and can be used for disease monitoring of osteoarthritis, but there is currently a lack of research on early warning serum markers for elderly osteoarthritis. In recent years, aptamers, as a single-stranded oligonucleotide (RNA / ssDNA) screened by systematic evolution of ligands by exponential enrichment (SELEX) technology, can bind to the target protein with high specificity, high stability and high affinity through its three-dimensional structure with the help of hydrogen bonds, hydrophobic bonds, van der Waals forces or ionic bonds.

[0003] Studies have confirmed that nucleic acid aptamers can successfully target ions, small molecules, peptides, proteins, cells and tissues. They can be used as detection reagents and imaging tools to quantify and track specific targets, and can also be used for targeted drug delivery, or even directly as a new type of biological drug to treat diseases. Given the title of nucleic acid aptamers as "chemical antibodies", they are often used as specific recognition elements in the design of sensors, including fluorescent sensors, biosensors and electrochemical sensors, to quickly detect target substances in complex sample matrices.

[0004] Osteoarthritis is the most common joint disease in the world, and early diagnosis is crucial to the patient's quality of life and disease progression. However, current diagnostic methods have many limitations, so we need more non-invasive and accurate biomarkers to assist in diagnosis. Cartilage acidic protein, as a new potential biomarker for osteoarthritis, has been widely used in disease monitoring. Studies have shown that CRTAC1 expression is significantly increased in the synovial fluid and serum of patients with osteoarthritis, which is closely related to the severity of the disease.

[0005] Based on the importance of cartilage acidic protein, we developed targeted aptamers for it. Aptamers, as "chemical antibodies", have the advantages of simple synthesis, low cost, high stability, and can recognize and bind to target molecules with high affinity and specificity. Through SELEX technology, we screened and optimized nucleic acid aptamers that specifically bind to CRTAC1.

[0006] These aptamers show great potential in the early diagnosis of osteoarthritis. They not only improve the specificity and sensitivity of diagnosis, but can also be used for real-time and quantitative detection of cartilage acidic protein, providing a new method for early diagnosis and monitoring of the disease. Compared with traditional biomarkers and imaging methods, aptamer detection has obvious advantages. Based on this, we have developed a serum detection kit based on nucleic acid aptamers to measure the content of CRTAC1 protein in the blood. The kit has high sensitivity and good specificity, which helps to achieve early intervention and improve the quality of life of patients. Summary of the invention

[0007] Technical problem solved: In view of the above technical problems, the present invention provides an early diagnosis kit for osteoarthritis and a construction method thereof, which are used to determine the CRTAC1 protein content in blood. The horseradish peroxidase (HRP)-labeled aptamers react with the sample to be tested in sequence, and the complementary chain (cDNA) of the aptamer is fixed on a microplate, and the CRTAC1 content is quickly determined by the color change based on the enzyme-catalyzed substrate. This sequential reaction avoids excessive binding of the aptamer to the cDNA, thereby ensuring full coverage recognition of CRTAC1. In the determination of plasma, CRTAC1 shows high sensitivity and stability, as well as good spike recovery rate.

[0008] Technical solution: In the first aspect, the present invention provides an early diagnosis kit for osteoarthritis, comprising an aptamer targeting cartilage acidic protein, an early biomarker of osteoarthritis, a capture probe and a recognition probe, wherein the nucleotide sequence of the aptamer is shown in SEQ ID NO.1; the capture probe is a reverse complementary strand of a biotin-modified aptamer, and its nucleotide sequence is shown in SEQ ID NO.2; the recognition probe is a biotin-modified aptamer, wherein: SEQ ID NO.1: 5′-ATACCAGCTTATTCAATTCAGCCGAAGCACGACGGGGTCCGCGAGTTCAGGTCTTTTCAGCTAAGCCAAGATAGTAAGTGCAATCT-3′; SEQ ID NO.2: 5′-AGATTGCACTTACTATCTTGGCTTAGCTGAAAAGACCTGAACTCGCGGACCCCGTCGTGCTTCGGCTGAATTGAATAAGCTGGTAT-3′.

[0009] Preferably, the kit further comprises streptomycin-modified horseradish peroxidase and a polystyrene microplate pre-blocked with streptavidin-coated BSA.

[0010] In a second aspect, the present invention provides a method for constructing the kit according to the first aspect, comprising the following steps: S1. Construction of capture probe: Add TE buffer to the SA-modified microplate to dilute the biotin-modified aptamer complementary cDNA chain and place it in a refrigerator at 4°C for later use; S2. Construction of recognition probe: dilute the SA-modified HRP to the required concentration, then mix it with the biotin-labeled aptamer at a volume ratio of 7:3 and place it in a refrigerator at 4°C for later use; S3. Operation of the kit: Mix the recognition probe with the plasma sample, add it to the microplate, incubate it with the capture probe, add 3,3',5,5'-tetramethylbenzidine solution for color development, add sulfuric acid to stop, and measure the final absorbance at 450 nm.

[0011] In a third aspect, the present invention provides use of the kit described in the first aspect or the kit constructed by the construction method described in the second aspect in monitoring the progression of osteoarthritis.

[0012] Preferably, the osteoarthritis is senile osteoarthritis.

[0013] Beneficial effects: The osteoarthritis early diagnosis kit established by the present invention is used to determine the CRTAC1 protein content in the blood. The horseradish peroxidase (HRP)-labeled aptamers are sequentially reacted with the sample to be tested, and the complementary chain (cDNA) of the aptamer is fixed on a microplate, and the CRTAC1 content is quickly determined by the color change based on the enzyme-catalyzed substrate. This sequential reaction avoids excessive binding of the aptamer to the cDNA, thereby ensuring full coverage recognition of CRTAC1. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is a schematic diagram of the principle and operation of an early diagnosis kit for osteoarthritis of the present invention; Figure 2 It is the distribution of aptamer lengths after high-throughput sequencing of the final library; Figure 3 It is a two-dimensional structural simulation of the nucleic acid aptamer SEQ ID NO.1; Figure 4 It is the prediction result of docking the aptamer SEQ ID NO.1 with cartilage acidic protein (CRTAC1) using an open source program; Figure 5 It is an analysis of the docking binding site between the aptamer SEQ ID NO.1 and the cartilage acidic protein (CRTAC1) molecule using an open source website service. DETAILED DESCRIPTION

[0015] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments: Example 1

[0016] A method for constructing an osteoarthritis early diagnosis kit comprises the following steps: S1. Analysis: S1-1, Cartilage acidic protein (CRTAC1) targeted aptamer sequencing The high-throughput sequencing technology was used to sequence and analyze the screened aptamer library, and a number of aptamers that can specifically bind to cartilage acidic protein were obtained. The sequences with high enrichment in the sequencing results were selected as candidate aptamers. The sequence length distribution is as follows: Figure 2 As shown, the sequence is concentrated at 50 bp, and the final sequence length is 86 bp, plus the universal sequences at both ends; S1-2. Bioinformatics analysis of candidate aptamers In order to further screen suitable aptamer sequences, a series of bioinformatics analyses were performed on the candidate aptamers, including secondary structure prediction using mfold and Gibbs free energy evaluation, which showed that the screened aptamer sequences had stable secondary structures and low Gibbs free energy, and the structures were stable, such as Figure 3 As shown; HADDOCK was used to predict the molecular docking of candidate aptamer ligands with cartilage acidic protein (CRTAC1), as shown Figure 4 As shown in Figure 2, it indicates that the screened nucleic acid aptamer sequence can stably bind to the target protein; the binding sites and bonding conditions are statistically analyzed, as shown in Figure 2. Figure 5 As shown in the figure: the strength and binding site of the aptamer sequences screened and the target protein were analyzed from the bonding conditions such as hydrogen bonds and salt bridges; finally, the potential aptamer sequences were screened out as shown in SEQ ID NO.1, SEQ ID NO.1: 5′-ATACCAGCTTATTCAATTCAGCCGAAGCACGACGGGGTCCGCGAGTTCAGGTCTTTTCAGCTAAGCCAAGATAGTAAGTGCAATCT-3′.

[0017] S2. Preparation of capture probe: First, SA was diluted in carbonate buffer (50 mM, pH 9.6), and 50 μL of SA solution was added to each well of a 96-well microplate and incubated for 1 hour at room temperature, which was controlled at about 25 °C. After that, the liquid in the well was poured out and washed three times with PBS-T buffer (1× PBS buffer containing 0.05% Tween-20) and the washing solution was poured out. In order to reduce nonspecific adsorption, the 96-well plate was blocked by adding 300 μL of BSA solution to the wells that were not completely coated with SA, and the wells were blocked for 1 h. Then, the wells were washed 3 times with PBS-T, and the washing solution was retained for 1 min each time. Finally, a SA-modified microplate was obtained.

[0018] The biotin-modified cDNA strand (5′-AGATTGCACTTACTATCTTGGCTTAGCTGAAAAGACCTGAACTCGCGGACCCCGTCGTGCTTCGGCTGAATTGAATAAGCTGGTAT-3′) was diluted with TE buffer (pH 8.0), and 50 μL was added to each well of the SA-modified microplate and incubated for 10 minutes. After that, the solution in the well was poured out and washed three times with PBS-T buffer and the washing solution was drained. The prepared capture probe was finally stored in a refrigerator at 4°C for later use.

[0019] S3. Preparation of identification probes: SA-modified HRP (1 mg / mL) was diluted to the desired concentration with PBS (pH 7.2) and then mixed with the biotin-labeled aptamer at a volume ratio of 7:3. The mixture was added to a centrifuge tube and incubated on a shaker for 30 min. Finally, the prepared recognition probe was stored in a refrigerator at 4 °C for later use.

[0020] S4. Operation of the kit: like Figure 1 As shown: First, the recognition probe was mixed with an equal volume of CRTAC1 sample at room temperature for about 5 minutes to obtain a mixture solution. Then, 100 μL of the mixture solution was loaded into each well of the microplate and incubated with the capture probe for about 30 minutes. The microplate was washed 3 times with PBS-T and the wash solution was drained. In the subsequent step, 100 μL of TMB solution was added to each well, and 50 μL of 2 M H2SO4 solution was added after 5 minutes to terminate the enzyme catalysis. Absorbance A 450 Scanned by Premium Reader, relative to A 450 Used to calculate the CRTAC1 content, which is obtained by the formula (Ab − As) / Ab, where Ab represents the blank A 450 Value, As represents the A of the sample 450 value.

[0021] Initially, the capture probe of biotin-labeled cDNA is anchored on the surface of streptavidin (SA)-coated and BSA-preblocked polystyrene microplate wells through the specific interaction between SA and biotin. The recognition probe is constructed through the coupling between SA-modified HRP and biotin-labeled aptamer. In the absence of CRTAC1 in the sample, there is no binding event between the aptamer and CRTAC1, so the recognition probe will subsequently hybridize with the capture probe through the base complementary pairing between the aptamer and cDNA. After washing, the abundant aptamer / biotin-SA / HRP complex remains in the well, where much HRP is available for the added TMB reporter gene to fully catalyze, resulting in a drastic color change from pale to dark yellow. In the presence of CRTAC1 in the sample, the aptamer element-attached recognition probes will first target the CRTAC1 analyte due to their specific recognition and high affinity binding, resulting in a reduction in the amount of unbound recognition probes. Correspondingly, there is less complex of aptamer / biotin-SA / HRP, resulting in insufficient TMB catalysis and varying degrees of color change, where the hue is negatively correlated with the CRTAC1 concentration in the sample. The presented kit is performed by adopting a convenient "mix-wash-detect" workflow and indirect competition based on sequential reactions, ensuring full coverage binding of CRTAC1 for robust assays and reducing accessibility to nonspecific binding sites on the surface. The results of the yellow shade are directly observed by naked eye and illuminated at 450nm (A 450 ) to measure the absorbance, thereby enabling semi-quantitative determination.

[0022] The kit of the present invention sequentially reacts horseradish peroxidase (HRP)-labeled aptamers with the sample to be tested, and fixes the complementary chain (cDNA) of the aptamer on a microplate, and rapidly determines the CRTAC1 content by the color change based on the enzyme-catalyzed substrate. This sequential reaction avoids excessive binding of the aptamer to the cDNA, thereby ensuring full coverage recognition of CRTAC1, and has a good effect in early warning of the occurrence and progression of osteoarthritis in the elderly.

[0023] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A kit for early diagnosis of osteoarthritis, characterized in that: It comprises an aptamer targeting cartilage acidic protein, an early biomarker of osteoarthritis, a capture probe and a recognition probe, wherein the nucleotide sequence of the aptamer is shown in SEQ ID NO.1; the capture probe is the reverse complementary chain of the biotin-modified aptamer, and its nucleotide sequence is shown in SEQ ID NO.2; and the recognition probe is the biotin-modified aptamer.

2. The osteoarthritis early diagnosis kit according to claim 1, characterized in that: Also included are polystyrene microplates pre-blocked with streptavidin-modified horseradish peroxidase and streptavidin-coated BSA.

3. A method for constructing the kit according to claim 1 or 2, characterized in that: The following steps are involved: S1. Construction of capture probe: Add TE buffer to the SA-modified microplate to dilute the biotin-modified aptamer complementary cDNA chain and place it in a refrigerator at 4°C for later use; S2. Construction of recognition probe: dilute the SA-modified HRP to the required concentration, then mix it with the biotin-labeled aptamer at a volume ratio of 7:3 and place it in a refrigerator at 4°C for later use; S3. Operation of the indirect competitive enzyme-linked aptamer assay kit: Mix the recognition probe with the plasma sample, add it to the microplate, incubate it with the capture probe, add 3,3',5,5'-tetramethylbenzidine solution for color development, add sulfuric acid to stop, and measure the final absorbance at 450nm.

4. Use of the kit according to claim 1 or 2 or the kit constructed by the construction method according to claim 3 in monitoring the progression of osteoarthritis.

5. The use according to claim 4, characterized in that: The osteoarthritis is senile osteoarthritis.

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