Detection biomarker of ocular fundus neovascular disease, detection kit and application

Abstract

The application belongs to the technical field of biomedical detection, and discloses a biomarker for detecting ocular fundus neovascular disease, a detection kit and application. The biomarker for detecting ocular fundus neovascular disease is tRNA-derived fragment tRF-1001, the nucleotide sequence of the biomarker for detecting ocular fundus neovascular disease is SEQ ID NO:1, and the detection primer of the biomarker for detecting ocular fundus neovascular disease is SEQ ID NO:4 and SEQ ID NO:5. The application uses real-time fluorescent quantitative PCR technology to detect the relative content of tRNA-derived fragment tRF-1001 in aqueous humor, and provides a new detection marker and prognosis evaluation method for ocular fundus neovascular disease. The application proves that it is completely feasible to realize the diagnosis of ocular fundus neovascular disease by detecting the expression of tRF-1001 in the aqueous humor of patients, and has the characteristics of small trauma and simple operation.

Description

Technical Field

[0001] This invention belongs to the field of biomedical detection technology, and in particular relates to a biomarker, detection kit, and application for detecting fundus neovascular diseases. Background Technology

[0002] Neovascular diseases of the fundus are a group of diseases characterized by pathological angiogenesis in the fundus. They can occur at different ages, such as retinopathy of prematurity (ROP) in newborns, proliferative diabetic retinopathy (PDR) in middle-aged adults, and age-related macular degeneration (AMD) in the elderly. They seriously threaten human vision and have become a global public health problem and socioeconomic burden. The important pathological features of these diseases are: damage to the vascular barrier, vascular occlusion and non-perfusion, pathological angiogenesis and secondary hemorrhage and leakage, which can lead to blindness.

[0003] Currently, the main treatments for this type of disease include systemic therapy, retinal laser photocoagulation, intravitreal injections of drugs, and vitrectomy. These treatments are primarily for advanced stages of the disease and all have significant limitations and potential side effects. As a non-destructive treatment, intravitreal injection of anti-VEGF drugs has become the mainstream anti-angiogenic therapy. However, some patients do not respond to anti-VEGF drug treatment and may even experience side effects such as retinal atrophy. Therefore, exploring the regulatory mechanisms underlying its pathogenesis and identifying sensitive and easily applicable diagnostic markers and novel therapeutic targets will contribute to the early diagnosis and prevention of retinal neovascularization diseases.

[0004] tRNA fragments are a group of non-coding RNAs that are produced by the nuclear cleavage of pre-tRNA and mature tRNA. They participate in the transcriptional and post-transcriptional regulation of gene expression, including ribosomal biogenesis, translational repression, and mRNA stability inhibition. Furthermore, tRNA fragments play important roles in many biological processes, including ribosomal biogenesis, epigenetic regulation, translational repression, and mRNA stability inhibition. tRNA fragments are involved in various human biological processes and diseases, and their abnormal regulation is significantly associated with diet-induced metabolic disorders and cancer progression. Given the characteristics and disease relevance of tRNA fragments, they hold promise as diagnostic biomarkers and prognostic markers.

[0005] Based on the above analysis, the problems and shortcomings of the existing technology are as follows:

[0006] (1) Early diagnosis of neovascular diseases of the fundus is crucial. However, existing technologies are difficult to make early specific diagnosis and lack minimally invasive early diagnostic methods.

[0007] (2) Current treatments for neovascular diseases of the fundus mainly target late-stage lesions, and all of them have many shortcomings and relative side effects.

[0008] (3) Intraocular injection of anti-VEGF drugs is currently the mainstream anti-angiogenic therapy, but some patients do not respond to anti-VEGF drug treatment or even experience side effects such as retinal atrophy. Summary of the Invention

[0009] To address the problems existing in the prior art, this invention provides a biomarker, a detection kit, and its application for detecting fundus neovascular diseases, particularly a biomarker, a detection kit, and its application based on real-time quantitative PCR technology for detecting fundus neovascular diseases.

[0010] The present invention is achieved as follows: a biomarker for detecting fundus neovascular disease, wherein the biomarker for detecting fundus neovascular disease is a tRNA-derived fragment tRF-1001, and the nucleotide sequence of the biomarker for detecting fundus neovascular disease is SEQ ID NO: 1.

[0011] Another objective of this invention is to provide a real-time quantitative PCR detection kit for fundus neovascularization using the aforementioned biomarkers for detecting fundus neovascularization. The kit includes a PCR amplification system comprising SYBR Premix Ex Taq 2×100μL, tRF-1001-specific qRT-PCR upstream primers, tRF-1001-specific qRT-PCR downstream primers, GAPDH quantitative PCR upstream primers, and GAPDH quantitative PCR downstream primers, one tube of each primer, 10μM, 100μL / tube.

[0012] Furthermore, the SYBR Premix Ex Taq 2× includes Ex Taq enzyme, dNTP Mixture, and Mg 2+ ,TliRNaseH,TB Green.

[0013] Furthermore, the upstream primer nucleotide sequence for the GAPDH quantitative PCR is SEQ ID NO: 2, and the downstream primer nucleotide sequence is SEQ ID NO: 3; the upstream primer nucleotide sequence for the tRF-1001-specific qRT-PCR is SEQ ID NO: 4, and the downstream primer nucleotide sequence is SEQ ID NO: 5; the random primers for RNA reverse transcription are the GAPDH quantitative PCR primer sequences.

[0014] Furthermore, the real-time fluorescence quantitative PCR detection kit for fundus neovascular diseases also includes a reverse transcription reaction system, which includes total RNA reverse transcription primers, 1 tube, 50 μM, 50 μL / tube; reverse transcriptase, 200 U / μL, 50 μL; dNTP Mixture, 10 mM each 50 μL; and reverse transcription buffer 50 μL.

[0015] Furthermore, the total RNA reverse transcription primers include Oligo dT and Random 6mers.

[0016] Furthermore, the real-time fluorescence quantitative PCR detection kit for fundus neovascular diseases also includes an RNA extraction system, which comprises: Trizol reagent, 1 tube, 2000 μL / tube; chloroform, 1 tube, 500 μL / tube; anhydrous ethanol, 1 tube, 8000 μL / tube; DEPC ddH2O, 1 tube, 1000 μL / tube; ddH2O, 1 tube, 2000 μL / tube; and isopropanol, 8000 μL / tube.

[0017] Another object of the present invention is to provide a method for screening biomarkers for detecting fundus neovascular disease, wherein the method comprises the following steps:

[0018] Step 1, Sample preparation: Collect aqueous humor samples from patients with wet age-related macular degeneration and patients with senile cataracts. RNA was extracted using TRIzol reagent and stored at -80℃ for later use.

[0019] Step 2: Aqueous humor samples from patients with wet age-related macular degeneration were used as the experimental group, and aqueous humor samples from patients with senile cataracts were used as the control group. Total RNA was reverse transcribed using a reverse transcription kit.

[0020] Step 3: The experimental results of the quantitative PCR chip analysis were verified to confirm the expression differences of target tRF-1001 in the aqueous humor of patients with wet age-related macular degeneration and cataracts.

[0021] Another object of the present invention is to provide the application of the aforementioned biomarker for detecting fundus neovascular disease in the preparation of diagnostic reagents for fundus neovascular disease.

[0022] Another objective of this invention is to provide the application of the aforementioned biomarker for detecting fundus neovascular disease in the preparation of a prognostic assessment reagent after treatment of fundus neovascular disease.

[0023] Based on the above technical solutions and the technical problems solved, please analyze the advantages and positive effects of the technical solution to be protected by this invention from the following aspects:

[0024] First, addressing the technical problems existing in the prior art and the difficulty in solving them, this paper closely analyzes, in conjunction with the technical solution to be protected by this invention and the results and data obtained during the research and development process, how the technical solution of this invention solves the technical problems, and the inventive technical effects brought about by solving these problems. The specific description is as follows:

[0025] This invention provides a biomarker, a detection kit, and applications for detecting fundus neovascular diseases. It discloses the expression of tRF-1001 in the aqueous humor of patients with cataracts and fundus neovascular diseases, including a method for detecting tRF-1001 by real-time quantitative PCR. This provides a biological basis for the diagnosis and prognostic assessment of fundus neovascular diseases and solves or at least alleviates some of the problems in the prior art.

[0026] This invention utilizes real-time quantitative PCR technology to detect the relative content of tRF-1001 in the aqueous humor, compare the differences in tRF-1001 content between normal individuals and patients under test, assess the disease susceptibility of patients under test, and provide a new detection marker and prognostic assessment method for fundus neovascular diseases.

[0027] This invention demonstrates that quantitative PCR technology is a mature and widely used detection technique, and the RNA extraction and reverse transcription processes can be performed using commercially available kits. Therefore, detecting tRF-1001 expression in the aqueous humor of patients to diagnose fundus neovascular diseases is entirely feasible.

[0028] The reagent kit provided by this invention has simple and clear operation steps, is convenient and quick to use, and has the characteristics of high sensitivity and minimal invasiveness.

[0029] The kit of the present invention is also applicable to the early screening of fundus neovascular disease: patients who are initially suspected of having fundus neovascular disease based on their age, medical history and local signs.

[0030] The kit of this invention was used to detect the tRF-1001 content in the aqueous humor of several patients with known fundus neovascularization and several cataract patients, respectively, as a standard. The same method was then used to determine the tRF-1001 content in the aqueous humor of unknown patients. The results were compared with the above standard data to preliminarily determine whether the patient had fundus neovascularization, in order to further confirm the diagnosis.

[0031] The kit of this invention is the first to utilize quantitative PCR to detect the tRF-1001 content in the aqueous humor to assist in the diagnosis and prognostic assessment of fundus neovascular diseases. It is characterized by minimal invasiveness and high operability, making tRF-1001 a biomarker that helps to scientifically determine the occurrence of fundus neovascular diseases and the prognosis of patients.

[0032] Second, considering the technical solution as a whole or from the perspective of the product, the technical effects and advantages of the technical solution to be protected by this invention are specifically described as follows:

[0033] This invention utilizes real-time quantitative PCR technology to detect the relative content of the tRNA-derived fragment tRF-1001 in the aqueous humor, providing a new detection marker and prognostic assessment method for fundus neovascular diseases. The kit provided by this invention is characterized by minimal invasiveness and simple operation.

[0034] Third, as supplementary evidence of the inventive step of the claims of this invention, it is also reflected in the following important aspects:

[0035] The expected benefits and commercial value of the technical solution of this invention after its commercialization are as follows: Currently, there is relatively little research in China on disease markers for detecting fundus neovascularization in the aqueous humor. This invention uses real-time quantitative PCR to analyze the expression of tRF-1001 in the aqueous humor of patients with wet age-related macular degeneration for disease diagnosis and prognosis, solving the problems of low specificity and poor sensitivity of previous kits for detecting fundus neovascularization. By combining this invention with clinical imaging examinations, it can significantly improve the detection efficiency of fundus neovascularization and provide early intervention targets and prognostic criteria, thus possessing significant commercial value. Attached Figure Description

[0036] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0037] Figure 1 This is a flowchart of a method for screening biomarkers for detecting fundus neovascular diseases provided in an embodiment of the present invention;

[0038] Figure 2 This is a graph showing the quantitative PCR detection results in the aqueous humor of patients with neovascularization of the fundus provided in Embodiment 1 of the present invention; the horizontal axis represents aqueous humor samples from different patients, and the vertical axis represents the expression level of tRF-1001;

[0039] Figure 3This is a schematic diagram of the quantitative PCR detection results of aqueous humor samples before and after treatment of fundus neovascular disease provided in Embodiment 2 of the present invention. The horizontal axis represents aqueous humor samples of fundus neovascular disease, and the vertical axis represents the expression level of tRF-1001. Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0041] To address the problems existing in the prior art, the present invention provides a biomarker, a detection kit, and its application for detecting fundus neovascular diseases. The present invention will be described in detail below with reference to the accompanying drawings.

[0042] I. Explanatory and Illustrative Embodiments. To enable those skilled in the art to fully understand how the present invention is specifically implemented, this section provides an explanatory and illustrative description of the embodiments described in the claims.

[0043] like Figure 1 As shown, the method for screening biomarkers for detecting fundus neovascular diseases provided in this embodiment of the invention includes the following steps:

[0044] S101, Sample preparation: Aqueous humor samples were collected from patients with wet age-related macular degeneration and patients with senile cataracts. RNA was extracted using TRIzol reagent and stored at -80℃ for later use.

[0045] S102, using aqueous humor samples from patients with wet age-related macular degeneration as the experimental group and aqueous humor samples from patients with senile cataracts as the control group, total RNA was reverse transcribed using a reverse transcription kit;

[0046] S103, using quantitative PCR to validate the results of chip analysis, verified the differential expression of target tRF-1001 in the aqueous humor of patients with wet age-related macular degeneration and cataracts.

[0047] This invention has determined that changes in tRF-1001 expression are significantly correlated with the occurrence of fundus neovascular diseases, and tRF-1001 was ultimately selected as a biomarker for the diagnosis of fundus neovascular diseases.

[0048] The biomarker for detecting fundus neovascular disease provided in this embodiment of the invention is the tRNA-derived fragment tRF-1001, and the nucleotide sequence of this biomarker is SEQ ID NO: 1.

[0049] The detection primers for the biomarkers of fundus neovascular disease provided in this embodiment of the invention are SEQ ID NO: 4 and SEQ ID NO: 5.

[0050] The real-time quantitative PCR detection kit for fundus neovascular diseases provided in this invention includes a PCR amplification system comprising SYBR Premix Ex Taq 2× (including Ex Taq enzyme, dNTP Mixture, Mg...) 2+ 100 μL of Tli RNase H (TB Green), tRF-1001-specific qRT-PCR upstream primer, tRF-1001-specific qRT-PCR downstream primer, GAPDH quantitative PCR upstream primer, GAPDH quantitative PCR downstream primer, 1 tube of each primer, 10 μM, 100 μL / tube.

[0051] The tRF-1001-specific qRT-PCR upstream primer provided in this embodiment of the invention is SEQ ID NO: 4, and the downstream primer is SEQ ID NO: 5; the GAPDH quantitative PCR upstream primer sequence is SEQ ID NO: 2, and the downstream primer sequence is SEQ ID NO: 3. The random primers for RNA reverse transcription are the GAPDH quantitative PCR primer sequences.

[0052] The real-time quantitative PCR detection kit for fundus neovascular diseases provided in this embodiment of the invention also includes a reverse transcription reaction system, which includes total RNA reverse transcription primers (including Oligo dT and Random 6mers), 1 tube, concentration: 50 μM, 50 μL / tube, reverse transcriptase (200 U / μL) 50 μL, dNTP Mixture (10 mM each) 50 μL, and reverse transcription buffer 50 μL.

[0053] The real-time fluorescence quantitative PCR detection kit for fundus neovascular diseases provided in this embodiment of the invention also includes an RNA extraction system, which includes Trizol reagent, 1 tube, 2000 μL / tube; chloroform, 1 tube, 500 μL / tube; anhydrous ethanol, 1 tube, 8000 μL / tube; DEPC ddH2O, 1 tube, 1000 μL / tube; ddH2O, 1 tube, 2000 μL / tube; and isopropanol, 8000 μL / tube.

[0054] The experimental steps provided in this embodiment of the invention for analyzing the feasibility of tRF-1001 as a prognostic agent for fundus neovascularization include:

[0055] Step 1, Sample preparation: Aqueous humor samples were collected from patients with wet age-related macular degeneration (experimental group) and patients with senile cataracts (control group). RNA was extracted using TRIzol (Invitrogen) reagent and stored at -80℃ for later use.

[0056] The second step involves reverse transcription of total RNA using a reverse transcription kit.

[0057] The third step involved using quantitative PCR to validate the experimental results analyzed by a microarray to verify the difference in expression of the target tRF-1001 in the aqueous humor of patients with wet age-related macular degeneration before and after treatment.

[0058] The RNA extraction and reverse transcription processes in these embodiments can be performed using commercially available kits. The technical solution of this invention will be further described below with reference to specific embodiments.

[0059] II. Application Examples. To demonstrate the inventiveness and technical value of the technical solution of this invention, this section provides application examples of the technical solution of the claims on specific products or related technologies.

[0060] Application Example 1: Application of tRF-1001 in the diagnosis of fundus neovascular diseases

[0061] Aqueous humor samples and processing: Aqueous humor samples were collected from 200 patients with wet age-related macular degeneration and matched from 200 patients with age-related cataracts as controls. Total RNA was extracted using TRIzol (Invitrogen) reagent and stored at -80℃ for later use.

[0062] Experimental procedure:

[0063] Step 1: Obtain the water sample to be tested.

[0064] The samples were aqueous humor samples from patients with neovascularization of the fundus and patients with senile cataracts.

[0065] Step 2: Extract RNA from the aqueous humor sample to be tested.

[0066] a) Transfer 0.03 mL of aqueous humor sample to a centrifuge tube, add 1 mL of Trizol, and use a pipette to repeatedly aspirate and swirl until the cells are completely lysed.

[0067] b) Add chloroform (1 / 5 the volume of sample solution + TRIzol Reagent), tighten the centrifuge tube cap, shake vigorously for 15 seconds, and let stand at room temperature for 5 minutes;

[0068] c) Centrifuge at 4℃, 12000g × 15min. Carefully remove the centrifuge tube from the centrifuge. At this point, the homogenate will be divided into three layers: a colorless supernatant, a white protein layer in the middle, and a colored organic phase in the lower layer. Transfer the supernatant to another new centrifuge tube (do not remove the white middle layer).

[0069] d) Add an equal volume of isopropanol to the supernatant and vortex to mix thoroughly;

[0070] e) Centrifuge at 4℃, 12000g × 10min. Generally, a precipitate will appear at the bottom of the test tube after centrifugation. Discard the supernatant.

[0071] f) Add 1 mL of 75% ethanol, gently invert by hand, centrifuge at 12000g for 5 min, and discard the supernatant;

[0072] g) Add 1 mL of anhydrous ethanol, gently invert by hand, centrifuge at 12000 g for 5 min, and discard the supernatant;

[0073] h) Dry at room temperature, add an appropriate amount of DEPC H2O to dissolve (promote dissolution at 65℃ for 10-15 min), and if necessary, gently blow the precipitate with a pipette;

[0074] i) BioDrop UV-Vis spectrophotometer measures RNA purity and concentration.

[0075] Step 3: Reverse transcribe the obtained RNA into cDNA.

[0076] Using TaKaRa's PrimeScript TM The RT reagent kit was used to prepare the reverse transcription reaction system according to the kit's specifications (25 μL): 5× PrimeScript TM Buffer 4μL, PrimeScript TM RT EnzymeMix I 1 μL, Oligo dT Primer (50 μM) 1 μL, Random 6mers (100 μM) 4 μL, total RNA 2 μL, RNase-free ddH2O 13 μL. Reverse to cDNA using the following program: 37℃ for 15 min, 85℃ for 5 sec. Store the resulting cDNA at -20℃.

[0077] Step 4, Real-time quantitative PCR

[0078] Take 50 μL of the PCR reaction system from the above kit and use TaKaRa's SYBR Premix Ex Taq. Prepare the system according to the instructions as follows: 25 μL of SYBR Premix Ex Taq, 1 μL each of the upstream and downstream primers for specific qRT-PCR (specifically recognizing tRF-1001 or GAPDH), 2 μL of cDNA from the sample to be tested, and add RNase-free ddH2O to a final volume of 50 μL.

[0079] PCR conditions:

[0080] 92℃ for 5 min, (92℃, 30 sec; 55℃, 30 sec; 72℃, 30 sec, 40 cycles), 72℃ for 5 min.

[0081] In this invention, the upstream primer for tRF-1001 detection is shown in SEQ ID NO: 4, and the downstream primer is shown in SEQ ID NO: 5; the upstream primer sequence for GAPDH quantitative PCR is shown in SEQ ID NO: 2, and the downstream primer sequence is shown in SEQ ID NO: 3.

[0082] Step 5, Data Analysis

[0083] Real-time quantitative PCR was performed on the samples to detect the target RNA and internal control RNA separately. The expression level of the target RNA was normalized based on the expression level of the internal control. Subsequently, the relative expression level of the target RNA was calculated using a standard method commonly used in this field. -△△ct Analysis: The Delta Ct value (ΔCt) of all aqueous humor samples was obtained by subtracting the Ct value of the target gene tRF-1001 from the Ct value of the internal reference gene GAPDH. This is expressed as ΔCt = Ct(target gene tRF-1001) - Ct(internal reference gene GAPDH). Then, the ΔCt value of the wet age-related macular degeneration group was subtracted from the ΔCt value of the senile cataract group to obtain the ΔΔCt value. This is expressed as ΔΔCt = ΔCt(wet age-related macular degeneration group) - ΔCt(senile cataract group). Finally, the relative expression level of the wet age-related macular degeneration group compared to the senile cataract group was calculated as 2. -△△ct The experiment was repeated three times, and the results were statistically analyzed. By comparing the expression differences of tRF-1001 in aqueous humor samples from patients with wet age-related macular degeneration and patients with senile cataracts, the susceptibility of the tested patients to the disease was determined.

[0084] Step Six, Result Judgment

[0085] The expression level of target tRF-1001 was detected by real-time quantitative PCR, and the ΔCt of patients with senile cataracts was determined (see Table 1). The ΔCt values ​​of the tested samples were compared to analyze the differences between patients with wet age-related macular degeneration (AMD) and senile cataracts. Further analysis was conducted to determine whether the values ​​of each sample were within the range for senile cataract patients. If the ΔCt of the tested sample was within or less than the ΔCt range for senile cataract patients, the sample was considered negative for wet AMD; if the ΔCt was greater than the ΔCt range, it was considered positive for wet AMD. (Based on 2...) -△△ct The analytical method showed that the relative expression level of the target gene was 2. -△△ct The result is as follows Figure 2 As shown. Figure 2 To analyze the expression difference of tRF-1001 in the aqueous humor of patients with wet age-related macular degeneration and senile cataract (50 typical cases selected from 200 cases in each group) using real-time quantitative PCR, the results showed that the expression of tRF-1001 in the aqueous humor of patients with wet age-related macular degeneration was significantly downregulated.

[0086] Table 1 ΔCt for each group of samples

[0087] Wet age-related macular degeneration Age-related cataracts tRF-1001Ct mean - GAPDH Ct mean 6.5±1.4 5.3±0.8

[0088] Based on the above results, aqueous humor samples were collected from 200 patients diagnosed with fundus neovascular disease by clinical imaging and 200 patients with senile cataracts, respectively, for analysis of tRF-1001 content in aqueous humor. The accuracy and sensitivity of detecting tRF-1001 content in aqueous humor were evaluated, and the results are shown in Table 2. The accuracy of aqueous humor samples was 88.3%, and the sensitivity was 90.0%. This indicates that tRF-1001 can be used as a biomarker for the diagnosis of fundus neovascular disease.

[0089] Table 2 Results of tRF-1001 as a biomarker for the diagnosis of fundus neovascular disease

[0090]

[0091] Application Example 2: Feasibility of using tRF-1001 as a prognostic marker

[0092] Step 1: Obtain the water sample to be tested.

[0093] Aqueous humor was collected from 200 patients with wet age-related macular degeneration before and after anti-angiogenic therapy.

[0094] Step 2: Extract RNA from the aqueous humor sample to be tested.

[0095] a) Transfer 0.03 mL of aqueous humor sample to a centrifuge tube, add 1 mL of Trizol, and use a pipette to repeatedly aspirate and swirl until the cells are completely lysed.

[0096] b) Add chloroform (1 / 5 the volume of sample solution + Trizol Reagent), tighten the centrifuge tube cap, shake vigorously for 15 seconds, and let stand at room temperature for 5 minutes;

[0097] c) Centrifuge at 4℃, 12000g × 15min. Carefully remove the centrifuge tube from the centrifuge. At this point, the homogenate will separate into three layers: a colorless supernatant, a white protein layer in the middle, and a colored organic phase in the lower layer. Transfer the supernatant to another new centrifuge tube (do not remove the white middle layer).

[0098] d) Add 1 volume of isopropanol to the supernatant and vortex to mix thoroughly;

[0099] e) Centrifuge at 4℃, 12000g × 10min. Generally, a precipitate will appear at the bottom of the test tube after centrifugation. Discard the supernatant.

[0100] f) Add 1 mL of 75% ethanol, gently invert by hand, centrifuge at 12000g for 5 min, and discard the supernatant;

[0101] g) Add 1 mL of anhydrous ethanol, gently invert by hand, centrifuge at 12000 g for 5 min, and discard the supernatant;

[0102] h) Dry at room temperature, add an appropriate amount of DEPC H2O to dissolve (promote dissolution at 65℃ for 10-15 min), and if necessary, gently blow the precipitate with a pipette.

[0103] i) BioDrop UV-Vis spectrophotometer measures RNA purity and concentration.

[0104] Step 3: The obtained RNA was reverse transcribed into cDNA using TaKaRa's PrimeScript. TM The RT reagent kit was prepared according to the provided 25 μL volume as follows: 5× PrimeScript TM Buffer 4μL, PrimeScript TM 1 μL of RT Enzyme Mix I, 1 μL of Oligo dT Primer (50 μM), 4 μL of Random 6mers (100 μM), 2 μL of total RNA, and 13 μL of RNase-free ddH2O. Reverse the DNA to cDNA using the following procedure: 37°C for 15 min, 85°C for 5 sec. Store the resulting cDNA at -20°C.

[0105] Step 4, Real-time quantitative PCR

[0106] The PCR reaction system (50 μL) was prepared according to the instructions of TaKaRa's SYBR Premix Ex Taq, using the following configuration: 25 μL of SYBR Premix Ex Taq, 1 μL each of the upstream and downstream primers for specific qRT-PCR (specifically recognizing tRF-1001 or GAPDH), 2 μL of cDNA from the sample to be tested, and RNase-free ddH2O to a final volume of 50 μL.

[0107] PCR conditions: 92℃, 5 min; 92℃, 30 sec; 55℃, 30 sec; 72℃, 30 sec, 40 cycles, 72℃, 5 min.

[0108] Step 5, Data Analysis

[0109] Gene expression values ​​are represented by 2 -△△ct The method was used to calculate the Delta Ct value (ΔCt) of all aqueous humor samples, assuming that the amplification efficiency of both the target gene and the reference gene was close to 100% and the relative deviation did not exceed 1 Ct. The Ct value of the target gene tRF-1001 was subtracted from the Ct value of the internal reference gene GAPDH from the Ct value of all aqueous humor samples, resulting in the Delta Ct value (ΔCt) of all aqueous humor samples. This was expressed by the formula ΔCt = Ct(target gene tRF-1001) - Ct(internal reference gene GAPDH). Then, the ΔCt value before treatment was subtracted from the ΔCt value before treatment to obtain the ΔΔCt value, expressed by the formula ΔΔCt = ΔCt(before treatment) - ΔCt(after treatment). Finally, the relative expression level of the wet age-related macular degeneration group after treatment compared to before treatment was calculated to be 2. -△△ct The results were statistically analyzed, and the treatment effect was judged by comparing the expression differences of tRF-1001 in aqueous humor samples before and after treatment in patients with wet age-related macular degeneration.

[0110] Step Six, Result Judgment

[0111] Calculate the 2 of the sample to be tested -△△ct The values ​​were analyzed to assess their changes before and after anti-angiogenic therapy. Results are as follows: Figure 3 As shown, aqueous humor was collected from patients with wet age-related macular degeneration before and after anti-angiogenic therapy; total RNA was extracted using Trizol reagent, and cDNA of the total RNA was obtained by reverse transcription PCR; the expression level of the target tRF-1001 was detected by real-time quantitative PCR (see figure). Figure 3 ). Figure 3To analyze the expression difference of tRF-1001 in the aqueous humor of patients with wet age-related macular degeneration before and after anti-angiogenic therapy (50 typical cases selected from 200 cases each), real-time quantitative PCR was used. The results showed that the expression of tRF-1001 in the aqueous humor of patients with wet age-related macular degeneration after receiving anti-angiogenic therapy was significantly upregulated, indicating that tRF-1001 may become a target for evaluating the efficacy and prognosis of anti-angiogenic therapy.

[0112] III. Evidence of the effects of the embodiments. The embodiments of the present invention have achieved some positive effects during the research and development or use process, and do indeed have great advantages compared with the prior art.

[0113] On the one hand, the expression difference of tRF-1001 in the aqueous humor of patients with wet age-related macular degeneration and senile cataract was analyzed by real-time quantitative PCR. The results showed that the expression of tRF-1001 in the aqueous humor of patients with wet age-related macular degeneration was significantly downregulated (see...). Figure 2 On the other hand, real-time quantitative PCR analysis of tRF-1001 expression in the aqueous humor of patients with wet age-related macular degeneration before and after anti-angiogenic therapy showed that tRF-1001 expression was significantly upregulated in the aqueous humor of patients with wet age-related macular degeneration after receiving anti-angiogenic therapy (see...). Figure 3 The above results suggest that tRF-1001 may be a target for evaluating the efficacy and prognosis of anti-angiogenic therapy.

[0114] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention, and within the spirit and principles of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. The application of a reagent for detecting biomarkers in the preparation of diagnostic reagents for wet age-related macular degeneration, characterized in that, The biomarker is a tRNA-derived fragment tRF-1001, and the nucleotide sequence of the biomarker is SEQ ID NO:

1.

2. A method for screening biomarkers for detecting wet age-related macular degeneration as described in claim 1, characterized in that, The method for screening biomarkers for wet age-related macular degeneration includes the following steps: Step 1, Sample preparation: Collect aqueous humor samples from patients with wet age-related macular degeneration and patients with senile cataracts. RNA was extracted using TRIzol reagent and stored at -80℃ for later use. Step 2: Aqueous humor samples from patients with wet age-related macular degeneration were used as the experimental group, and aqueous humor samples from patients with senile cataracts were used as the control group. Total RNA was reverse transcribed using a reverse transcription kit. Step 3: The experimental results of the quantitative PCR chip analysis were verified to confirm the expression differences of target tRF-1001 in the aqueous humor of patients with wet age-related macular degeneration and cataracts.

3. The application of the reagent for detecting biomarkers as described in claim 1 in the preparation of diagnostic reagents for wet age-related macular degeneration, characterized in that, The biomarker is a tRNA-derived fragment tRF-1001, and the nucleotide sequence of the biomarker is SEQ ID NO: 1.

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