阅读说明：本技术 一种糖尿病视网膜病变检测生物标记物、检测试剂盒及应用 (Biomarker for detecting diabetic retinopathy, detection kit and application ) 是由 颜标 李秀苗 蒋沁 朱君雅 孙亚男 姚进 姚牧笛 钟宇玲 于 2020-08-06 设计创作，主要内容包括：本发明属于生物医学检测技术领域,尤其涉及一种糖尿病视网膜病变检测生物标记物、检测试剂盒及应用。本发明利用实时荧光定量PCR技术,通过检测血清中LncRNA AQP4-AS1的相对含量,为糖尿病视网膜病变提供了新的检测标记物和预后评估方法,本发明提供的试剂盒具有创伤性小和操作简单等特点。(The invention belongs to the technical field of biomedical detection, and particularly relates to a biomarker for detecting diabetic retinopathy, a detection kit and application. The invention provides a novel detection marker and a prognosis evaluation method for diabetic retinopathy by detecting the relative content of LncRNA AQP4-AS1 in serum by utilizing a real-time fluorescent quantitative PCR technology, and the kit provided by the invention has the characteristics of small wound, simple operation and the like.)

1. A biomarker for detecting diabetic retinopathy based on a real-time fluorescent quantitative PCR technology is LncRNA AQP4-AS1, and the nucleotide sequence is shown AS SEQ ID No. 1.

2. The biomarker for detecting diabetic retinopathy based on the real-time fluorescent quantitative PCR technology as claimed in claim 1, wherein: the detection primers of the markers are shown as SEQ ID NO.4 and SEQ ID NO. 5.

3. The real-time fluorescence quantitative PCR detection kit for diabetic retinopathy is characterized by comprising the following components in parts by weight: the kit comprises a PCR amplification system, wherein the PCR amplification system comprises SYBR Premix Ex Taq 2 x, a qRT-PCR upstream primer specific to AQP4-AS1, a qRT-PCR downstream primer specific to AQP4-AS1, a GAPDH quantitative PCR upstream primer and a GAPDH quantitative PCR downstream primer.

4. The real-time fluorescent quantitative PCR detection kit for diabetic retinopathy according to claim 3, characterized in that: the specific qRT-PCR upstream primer of AQP4-AS1 is shown AS SEQ ID NO.4, and the specific downstream primer is shown AS SEQ ID NO. 5; the upstream primer sequence of GAPDH quantitative PCR is shown as SEQ ID NO. 2, and the downstream primer sequence is shown as SEQ ID NO. 3.

5. The real-time fluorescent quantitative PCR detection kit for diabetic retinopathy according to claim 3, characterized in that: the kit also comprises a reverse transcription reaction system, wherein the reverse transcription reaction system comprises Oligo dT and Random6mers, reverse transcriptase, dNTP mix and reverse transcription buffer.

6. The real-time fluorescent quantitative PCR detection kit for diabetic retinopathy according to claim 5, characterized in that: the kit also comprises an RNA extraction system, wherein the RNA extraction system comprises Trizol reagent, chloroform, absolute ethyl alcohol and DEPC ddH₂O，ddH₂O, isopropanol.

7. The use of the biomarker of claim 1 or 2 for the preparation of a diagnostic reagent for diabetic retinopathy, in particular for the detection of diabetic retinopathy based on real-time fluorescent quantitative PCR technology.

8. Use of the biomarker according to claim 1 or 2 for the preparation of a reagent for the prognosis evaluation after the treatment of diabetic retinopathy, in particular for the preparation of a reagent for the prognosis evaluation after the treatment of diabetic retinopathy based on the real-time fluorescence quantitative PCR technique.

Technical Field

The invention belongs to the technical field of biomedical detection, and particularly relates to a biomarker for detecting diabetic retinopathy, a detection kit and application.

Background

Diabetic Retinopathy (DR), one of the common complications of diabetes and one of the major blinding eye diseases, has increased year by year and is now a global public health problem and socioeconomic burden. Retinal vascular damage occurs early in the onset of DR and is manifested by a breakdown of the blood retinal barrier, increased vascular permeability, endothelial cell proliferation, pericyte loss, thickening of the basement membrane and the formation of new blood vessels. Complications such as vitreous hemorrhage, macular ischemia edema, retinal detachment and the like can occur at the later stage of the disease process, so that the visual function is seriously damaged.

At present, the treatment modes of diabetic retinopathy mainly comprise three main types of laser, medicine and operation. Laser treatments include whole retinal photocoagulation, localized photocoagulation, and grid photocoagulation. Local drug intervention means include glucocorticoid therapy and intravitreal injection of anti-vascular endothelial growth factor; the operation treatment is mainly vitreous cutting. However, none of these methods can fundamentally solve the problems of non-perfusion of the microvasculature and ischemia and hypoxia, and have limitations and side effects of treatment. Therefore, the method finds a sensitive detection marker which is convenient to apply aiming at the diagnosis and intervention of the diabetic retinopathy, and has very important clinical significance.

Long non-coding RNA (LncRNA) is a class of RNA molecules with a length of more than 200nt, and can regulate gene expression at various levels, such as epigenetic regulation, transcriptional regulation and post-transcriptional regulation. LncRNA is involved in a variety of important physiological processes such as genomic imprinting, chromosome modification, transcriptional activation, transcriptional interference, and nuclear transport. Numerous studies have demonstrated that LncRNAs play important regulatory roles in a variety of human diseases, including ophthalmic diseases, cardiovascular diseases, neurodegenerative diseases, and cancer. In view of the characteristics and disease relevance of IncRNA, it is expected to be a marker for diagnosis and a marker for prognosis evaluation.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a biomarker for detecting diabetic retinopathy, a detection kit and application thereof, discloses the expression conditions of LncRNA AQP4-AS1 in the serum of normal people and diabetic retinopathy patients, and provides a biological basis for the diagnosis and prognosis evaluation of diabetic retinopathy by a method for detecting LncRNA AQP4-AS1 by real-time fluorescence quantitative PCR (polymerase chain reaction), and aims to solve a part of problems in the prior art or at least alleviate a part of problems in the prior art.

The biomarker for detecting diabetic retinopathy based on the real-time fluorescent quantitative PCR technology is LncRNA AQP4-AS1, and the nucleotide sequence is shown AS SEQ ID No. 1.

Further, detection primers of the marker are shown as SEQ ID NO.4 and SEQ ID NO. 5.

The real-time fluorescence quantitative PCR detection kit for diabetic retinopathy is characterized by comprising the following components in parts by weight: comprises a PCR amplification system, wherein the PCR amplification system comprises SYBR Premix Ex Taq 2 x (comprising Ex Taq enzyme, dNTP mix, Mg2+, Tli RNaseH, TB Green)100 muL, AQP4-AS1 specific qRT-PCR upstream primer, 1 tube, 10 muM, 100 muL/tube, AQP4-AS1 specific qRT-PCR downstream primer, 1 tube, 10 muM, 100 muL/tube, GAPDH quantitative PCR upstream primer, 1 tube, 10 muM, 100 muL/tube, GAPDH quantitative PCR downstream primer, 1 tube, 10 muM, 100 muL/tube.

Further, the specific qRT-PCR upstream primer of AQP4-AS1 is shown AS SEQ ID NO.4, and the specific qRT-PCR downstream primer is shown AS SEQ ID NO. 5; the upstream primer sequence of GAPDH quantitative PCR is shown as SEQ ID NO. 2, and the downstream primer sequence is shown as SEQ ID NO. 3. The RNA reverse transcription random primer is GAPDH quantitative PCR primer sequence.

Further, the kit further comprises a reverse transcription reaction system, wherein the reverse transcription reaction system comprises total RNA reverse transcription primers (comprising Oligo dT and Random 6mers), 1 tube, concentration: 50 μ M, 50 μ L/tube, 50 μ L reverse transcriptase (200U/μ L), 50 μ L dNTP mix (10mM each), 50 μ L reverse transcription buffer.

The method specifically comprises the following steps: 5 XPrimeScript^TMBuffer，PrimeScript^TMRT Enzyme Mix I、Oligo dTPrimer(50μM)、Random 6mers(100μM)，RNase free ddH₂O。

Further, the kit also comprises an RNA extraction system, wherein the RNA extraction system comprises Trizolreagent, 1 tube and 2000 mu L/tube; chloroform, 1 tube, 500 μ L/tube; absolute ethyl alcohol, 1 tube, 8000 mul/tube; DEPC ddH₂O, 1 tube, 1000. mu.L/tube; ddH₂O, 1 tube, 2000. mu.L/tube; isopropanol, 8000 μ L/tube.

The application of the biomarker in preparing a diabetic retinopathy diagnostic reagent. In particular to the application in the preparation of the diagnostic reagent for detecting the diabetic retinopathy based on the real-time fluorescent quantitative PCR technology.

The biomarker is applied to the preparation of a prognosis evaluation reagent after the treatment of diabetic retinopathy, in particular to the preparation of a prognosis evaluation reagent after the treatment of diabetic retinopathy based on a real-time fluorescence quantitative PCR technology.

Δ Ct range for normal patients: 4.2-6.1. If the delta Ct of the sample to be detected is within the delta Ct range of the normal person or is larger than the delta Ct range, the sample to be detected is considered as a negative patient; if the delta Ct is less than the delta Ct range, the patient is considered positive.

The invention determines that the change of the expression level of LncRNA AQP4-AS1 has obvious correlation with the occurrence of diabetic retinopathy. And finally, the LncRNA AQP4-AS1 is selected AS a biomarker for diagnosing diabetic retinopathy. The method comprises the following steps:

the first step is as follows: sample preparation: serum samples of the proliferative diabetic retinopathy patients and serum samples of normal persons were collected, RNA was extracted using TRIzol (Invitrogen) reagent, and stored at-80 ℃ for later use.

The second step is that: carrying out reverse transcription of total RNA by adopting a reverse transcription kit;

the third step: the experimental result of chip analysis is verified by adopting quantitative PCR, and the expression difference of the target LncRNA AQP4-AS1 in the serum of patients with diabetic retinopathy and normal persons is verified.

It is another object of the present invention to analyze the feasibility of AQP4-AS1 AS an assessment of the prognostic efficacy of diabetic retinopathy. The experimental steps include:

the first step is as follows: sample preparation: serum samples of patients with proliferative diabetic retinopathy before and after treatment were collected, RNA was extracted using TRIzol (Invitrogen) reagent, and stored at-80 ℃ for future use.

The second step is that: carrying out reverse transcription of total RNA by adopting a reverse transcription kit;

the third step: the expression difference of the target LncRNA AQP4-AS1 in the serum of patients with diabetic retinopathy before and after treatment is verified by adopting the experimental result of chip analysis of quantitative PCR verification.

Lnc RNA AQP4-AS1 can be used AS antisense long-chain non-coding RNA of aquaporin 4(AQP4) and can regulate the expression of AQP 4. AQP4-AS1 is expressed primarily at the terminal ends of retinal Muller cells. AQP4-AS1 can regulate the function of Muller cells by regulating the expression of AQP4, indirectly influence the functions of endothelial cells and retinal ganglion cells, and play an important role in diabetic retinal blood vessels and neuropathy.

In summary, the advantages and positive effects of the invention are:

the invention provides a novel detection marker and a prognosis evaluation method for diabetic retinopathy by detecting the relative content of LncRNA AQP4-AS1 in serum by utilizing a real-time fluorescent quantitative PCR technology, and the kit provided by the invention has the characteristics of high sensitivity, small wound, simple operation and the like.

The invention proves that the diagnosis of the diabetic retinopathy is completely feasible by detecting the expression of LncRNA AQP4-AS1 in the serum of a patient through a quantitative PCR technology.

The kit of the invention is also suitable for: patients suspected of diabetic retinopathy were initially determined by age, history, and local signs.

The kit of the invention is used for respectively detecting the content of LncRNA AQP4-AS1 in the serum of a plurality of known diabetic retinopathy patients and a plurality of normal patients, and the content is used AS a standard. And determining the content of LncRNA AQP4-AS1 in the serum of an unknown patient by the same method, comparing the standard data, judging whether the content is in a corresponding numerical range, taking the value AS intermediate data (a direct diagnosis conclusion cannot be made according to the data), and further judging the condition of the patient by combining other detection data.

The kit disclosed by the invention is used for the first time, the quantitative PCR method is utilized, the diagnosis and prognosis evaluation of the diabetic retinopathy are assisted by detecting the content of LncRNA AQP4-AS1 in serum, and the kit has the characteristics of small wound and strong operability, so that the LncRNA AQP4-AS1 becomes a biomarker, and the scientific judgment on the occurrence of the diabetic retinopathy and the prognosis of a patient is facilitated.

Drawings

FIG. 1 is the result of quantitative PCR assay in serum of diabetic retinopathy patient of example 1; the abscissa represents serum samples of different patients, and the ordinate represents the expression level of LncRNA AQP4-AS 1;

FIG. 2 is the results of quantitative PCR assay of serum samples before and after treatment of diabetic retinopathy in example 2, the abscissa represents the diabetic retinopathy serum samples, and the ordinate represents the expression level of LncRNA AQP4-AS 1.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples, and the equipment and reagents used in the examples and test examples are commercially available without specific reference. The specific embodiments described herein are merely illustrative of the invention and are not intended to be limiting.

Various modifications to the precise description of the invention will be readily apparent to those skilled in the art from the information contained herein without departing from the spirit and scope of the appended claims. It is to be understood that the scope of the invention is not limited to the procedures, properties, or components defined, as these embodiments, as well as others described, are intended to be merely illustrative of particular aspects of the invention. Indeed, various modifications of the embodiments of the invention which are obvious to those skilled in the art or related fields are intended to be covered by the scope of the appended claims.

For a better understanding of the invention, and not as a limitation on the scope thereof, all numbers expressing quantities, percentages, and other numerical values used in this application are to be understood as being modified in all instances by the term "about". Accordingly, unless expressly indicated otherwise, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. In the present invention, "about" means within 10%, preferably within 5% of a given value or range.

The normal temperature in the following embodiments of the present invention refers to a natural room temperature condition in four seasons, and is not subjected to additional cooling or heating treatment, and is generally controlled at 10 to 30 ℃, preferably 15 to 25 ℃.

The invention discloses a biomarker for detecting diabetic retinopathy, a detection kit and application thereof, and particularly relates to the following embodiments. The processes of RNA extraction, reverse transcription and the like in the invention can be operated by referring to a kit purchased in the market.