Reagent compositions, kits and their use for detection of diabetic retinopathy

Abstract

The application provides a detection reagent composition and kit for diabetic retinopathy, and belongs to the field of molecular detection. The reagent composition in the application is used for detecting miRNA markers in blood samples to achieve the purpose of diagnosing diabetic retinopathy. The reagent composition comprises primers and buffer for RT-PCR. The buffer comprises Tris-HCl, NH4Cl, 1,2-propanediol, potassium phosphate and MgCl2. The molar concentration ratio of the components is 3-6:2-4:20-30:5-8:0.1-0.4. The kit provided by the application has good repeatability and sensitivity, and is suitable for application in clinical practice.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of molecular detection, and particularly relates to a reagent and a reagent composition for detecting diabetic retinopathy. BACKGROUND

[0002] Diabetic retinopathy (DR) is one of the most common microvascular complications of diabetes, and is a chronic and progressive complication of diabetes that causes retinal microvascular leakage and obstruction, thereby causing a series of ocular fundus lesions such as microaneurysms, hard exudation, cotton wool spots, neovascularization, vitreous proliferation, macular edema, and even retinal detachment. DR is divided into proliferative diabetic retinopathy and non-proliferative diabetic retinopathy according to whether there are abnormal neovascularization from the retina.

[0003] In the prior art, the detection of diabetic retinopathy is mostly performed by overall examination of the anterior segment to the fundus, that is, detailed examination of basic vision, intraocular pressure, cornea, iris, lens, fundus, and the like. In clinical practice, the anterior segment is first examined for abnormalities, because diabetic retinopathy can affect the anterior segment, and in the late stage, neovascular glaucoma can occur, and neovascularization can be seen on the iris. The most commonly used examination method for the fundus is mydriatic fundus examination. At present, devices such as wide-angle cameras and non-mydriatic cameras can be used for fundus examination and screening without mydriasis. The most classic examination method in clinical practice is fundus fluorescein angiography, which can be divided into two types. One is fundus fluorescein angiography (FFA), and the contrast agent is sodium fluorescein. The other is choroidal angiography, which is called ICG, and the contrast agent is indocyanine green. This examination can be used as the gold standard for the diagnosis of diabetic retinopathy.

[0004] However, the examination method in the prior art needs to rely on the judgment experience of a certain clinician on the one hand, and on the other hand, it is based on the symptoms of the disease, and when it can effectively diagnose, the patient has often been ill for a period of time, and it is difficult to perform early diagnosis. Detection by modern molecular technology has become the main research direction for early diagnosis of DR.

[0005] A serum / plasma miRNA marker associated with type 2 diabetic retinopathy and application is disclosed in Chinese patent application CN201710584620.8. The application discloses that the serum / plasma miRNA marker associated with type 2 diabetic retinopathy is one or a combination of more than two of hsa-let-7a-5p, hsa-miR-novel-chr5_15976, hsa-miR-28-3p, hsa-miR-20a-5p, hsa-miR-151a-5p, hsa-miR-148a-3p and hsa-miR-223-3p. The application provides a thought that miRNA is used as a diagnostic marker for type 2 diabetic retinopathy, and the marker itself is stable, can assist in diagnosing whether a diabetic patient is complicated with DR in the early stage, and can dynamically monitor the disease progression of a diabetic retinopathy patient. It can be applied to the preparation of a type 2 diabetic retinopathy early diagnosis product. However, the detection result of the amplification system is not stable, and the sensitivity is not very ideal in actual application, and there is a space for further optimization. SUMMARY

[0006] To solve the above problems, the application provides a reagent and a reagent composition for detecting diabetic retinopathy.

[0007] In one aspect, the application provides a reagent composition for detecting a miRNA marker in a blood sample.

[0008] The detection reagent comprises primers for detecting the miRNA marker.

[0009] The detection reagent further comprises a PCR buffer, wherein the PCR buffer comprises Tris-HCl, NH4Cl, 1,2-propanediol, potassium phosphate and MgCl2; and the molar concentration ratio of each component is 3-6:2-4:20-30:5-8:0.1-0.4.

[0010] Preferably, the molar concentration ratio of each component in the PCR buffer is 4-6:3-4:20-25:6-8:0.1-0.3.

[0011] Preferably, the molar concentration ratio of each component in the PCR buffer is 5:3:20:6:0.2.

[0012] Preferably, the use concentration of Tris-HCl is 30-60 mM, the use concentration of NH4Cl is 20-40 mM, the use concentration of 1,2-propanediol is 200-300 mM, the use concentration of potassium phosphate is 50-80 mM, and the use concentration of MgCl2 is 1-4 mM.

[0013] Further preferably, the concentration of Tris-HCl used is 40-60 mM, the concentration of NH4Cl used is 30-40 mM, the concentration of 1,2-propanediol used is 200-250 mM, the concentration of potassium phosphate used is 60-80 mM, and the concentration of MgCl2 used is 1-3 mM.

[0014] In some embodiments, the concentration of Tris-HCl used is 50 mM, the concentration of NH4Cl used is 30 mM, the concentration of 1,2-propanediol used is 200 mM, the concentration of potassium phosphate used is 60 mM, and the concentration of MgCl2 used is 2 mM.

[0015] The miRNA markers are hsa-let-7a-5p, hsa-miR-28-3p, hsa-miR-151a-5p, and hsa-miR-223-3p.

[0016] The sequence of hsa-let-7a-5p is SEQ ID No. 1, the sequence of hsa-miR-28-3p is SEQ ID No. 2, the sequence of hsa-miR-151a-5p is SEQ ID No. 3, and the sequence of hsa-miR-223-3p is SEQ ID No. 4.

[0017] The detection primer sequences are as follows:

[0018]

[0019] The detection reagent further comprises DNA polymerase, dNTPs, and ddH2O.

[0020] In another aspect, the present application provides a kit comprising the aforementioned reagent.

[0021] The kit can further comprise an RNA extraction reagent and / or a reverse transcription reagent.

[0022] The RNA extraction reagent and / or the reverse transcription reagent can be a reagent disclosed in the prior art or a reagent not yet disclosed, and the purpose is to obtain template DNA and then apply it to the PCR amplification system of the present application, so these reagents combined with the present application are within the scope of protection of the present application.

[0023] Advantages of the present application:

[0024] When the kit of the present application is used to detect diabetic retinopathy, it has good repeatability, high sensitivity, and can effectively improve the accuracy of the detection results. In the actual sample detection, it also shows the expected effect and has good clinical application prospects. DETAILED DESCRIPTION

[0025] The application will be further described in conjunction with specific examples. The following examples are not intended to limit the application, but merely to illustrate the application. Unless otherwise stated, the experimental procedures used in the following examples were carried out according to conventional conditions. Unless otherwise stated, the materials and reagents used in the following examples were obtained from commercial suppliers such as Sigma-Aldrich (St. Louis, MO, USA) and used according to the manufacturer's instructions.

[0026] In the following examples, the positive quality control was a synthetic cDNA fragment of hsa-let-7a-5p, hsa-miR-28-3p, hsa-miR-151a-5p and hsa-miR-223-3p. The negative quality control was deionized water.

[0027] In the following examples, the RNA extraction kit was Blood RNA Kit (YISAN, Cat. No. 19241ES50) and the reverse transcription kit was miRNA 1st Strand cDNA Synthesis Kit (by stem-loop) (Novogene, Cat. No. MR101).

[0028] Example 1: A kit for detecting diabetic retinopathy

[0029] The kit of this example comprises the following components:

[0030]

[0031]

[0032] Example 2: A method for detecting diabetic retinopathy

[0033] The sample blood was 250 μL, and the RNA was extracted by the kit. After the RNA quality was detected to be qualified, a commercially available kit was used to perform reverse transcription on the RNA to obtain cDNA for the next step of RT-PCR.

[0034] In this example, the concentration of Tris-HCl was 500 mM, the concentration of NH4Cl was 500 mM, the concentration of 1,2-propanediol was 1 M, the concentration of potassium phosphate was 1 M, and the concentration of MgCl2 was 20 mM.

[0035] All cDNA samples were prepared into PCR reaction systems as follows:

[0036]

[0037]

[0038] PCR reaction program as follows: 95℃, 10 min; 40 PCR cycles (95℃, 10 s; 60℃, 60 s (collect fluorescence)); to establish the melting curve of PCR product, after the amplification reaction, (95℃, 10 s; 60℃, 60 s; 95℃, 15 s); and from 60℃ slowly heated to 99℃ (the instrument automatically Ramp Rate is 0.05℃ / s);

[0039] The target miRNA and internal reference RNU6B (the reverse transcription primer and quantitative reverse primer thereof are SEQ ID No. 14, and the quantitative forward primer is SEQ ID No. 15) of each sample were subjected to Real-time PCR reaction respectively; the data was 2 -ΔΔ CT method was used for analysis.

[0040] The result value was interpreted according to the following:

[0041] High risk Medium risk Low risk hsa-let-7a-5p ＞2.80 2.00-2.80 ＜2.00 hsa-miR-28-3p ＞2.00 1.50-2.00 ＜1.50 hsa-miR-151a-5p ＞3.00 2.30-3.00 ＜2.30 hsa-miR-223-3p ＞2.30 1.30-2.30 ＜1.30

[0042] When all the test results fall into the low risk interval, it is determined that the diabetic retinopathy is low risk; if any test result falls into the high risk interval, it is determined that the diabetic retinopathy is high risk; if any test result falls into the medium risk interval, but no result falls into the high risk interval, it is determined that the diabetic retinopathy is medium risk.

[0043] Example 3

[0044] The difference from Example 2 is that the PCR reaction system is as follows:

[0045]

[0046]

[0047] Example 4

[0048] The difference from Example 2 is that the PCR reaction system is as follows:

[0049] Reagent Amount Tris-HCl 0.8 μL NH4CI 0.4 μL 1,2-propanediol 2.5 μL Potassium phosphate 0.8 μL MgCl2 0.5 μL DNA Polymerase 1 μL EvaGreen 1 μL Primer F / R (10 μM) 0.5 μL each cDNA 2 μL ddH2O qsp 10 μL

[0050] Performance detection of kit in experimental example 1

[0051] The performance of the kit provided in Example 1 was detected, and the methods of Examples 2-4 were referred to for detection.

[0052] (1) Reproducibility

[0053] The above kits were respectively used to detect the samples with the concentration of 10 copies / μL (low concentration) and 10 6Five tests were performed on each of the high-concentration positive control samples (copies / μL). The CV value was calculated to assess the repeatability of the kit. The results are as follows:

[0054]

[0055]

[0056] (2) Sensitivity

[0057] Dilute the positive control sample to different concentrations: 10 6 copies / μL, 10 5 copies / μL, 10 4 copies / μL, 10 3 copies / μL, 10 2 The limits of detection for each kit were measured at 10 copies / μL and 1 copy / μL, respectively, using the methods described in Examples 2-4. The results showed that the limits of detection for each kit were:

[0058] Example 2 Example 3 Example 4 Limit of detection 1 copies / μL 10 copies / μL 10 copies / μL

[0059] Experimental Example 2: Validation of Clinical and Irrelevant Samples

[0060] Blood samples from 10 patients with diabetic retinopathy (DR), 10 patients with diabetes but no DR, and 20 healthy volunteers (volunteers without DR or diabetes) from the First Affiliated Hospital of Wenzhou Medical University were tested using the method described in Example 2. The results were verified using the method described in Example 2.

[0061]

[0062]

[0063] The results showed that this kit can effectively distinguish between DR samples, diabetic samples without DR, and healthy samples, which is of great guiding significance for the detection of diabetic retinopathy.

[0064] Comparative Example

[0065] The comparative example is set up with reference to Example 2, as follows:

[0066] Comparative Example Difference from Example 2 Comparative Example 1 Buffer system without addition of potassium phosphate Comparative Example 2 No MgCl2 added to buffer system Comparative Example 3 Buffer system without NH4Cl Comparative Example 4 NH4CI is replaced by Tris-HCl

[0067] Following the methods of Experimental Examples 1 and 2, the PCR systems of Comparative Examples 1-4 were validated, and the repeatability results are as follows:

[0068]

[0069] Sensitivity results were as follows:

[0070] Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Limit of detection 10 2 copies / μL 10 3 copies / μL 10 3 copies / μL 10 2 copies / μL

Claims

1. A reagent composition for detecting miRNA markers in a blood sample, comprising primers and buffers for RT-PCR, characterized in that, The buffer comprises Tris-HCl, NH4Cl, 1,2-propanediol, potassium phosphate and MgCl2, and the molar concentration ratio of each component is 3-6:2-4:20-30:5-8:0.1-0.

4.

2. The reagent composition according to claim 1, characterized by The molar concentration ratio of Tris-HCl, NH4Cl, 1,2-propanediol, potassium phosphate and MgCl2 in the buffer is 4-6:3-4:20-25:6-8:0.1-0.

3.

3. The reagent composition of claim 2, wherein, The molar concentration ratio of Tris-HCl, NH4Cl, 1,2-propanediol, potassium phosphate and MgCl2 in the buffer is 5:3:20:6:0.

2.

4. The reagent composition of claim 1, wherein The use concentration of Tris-HCl is 30-60mM, the use concentration of NH4Cl is 20-40mM, the use concentration of 1,2-propanediol is 200-300mM, the use concentration of potassium phosphate is 50-80mM, and the use concentration of MgCl2 is 1-4mM.

5. The reagent composition according to claim 4, wherein The use concentration of Tris-HCl is 40-60mM, the use concentration of NH4Cl is 30-40mM, the use concentration of 1,2-propanediol is 200-250mM, the use concentration of potassium phosphate is 60-80mM, and the use concentration of MgCl2 is 1-3mM.

6. The reagent composition according to claim 5, wherein The use concentration of Tris-HCl is 50mM, the use concentration of NH4Cl is 30mM, the use concentration of 1,2-propanediol is 200mM, the use concentration of potassium phosphate is 60mM, and the use concentration of MgCl2 is 2mM.

7. The reagent composition of claim 1, wherein, The primer is used for amplifying the miRNA marker.

8. The reagent composition of claim 7, wherein, The miRNA marker is hsa-let-7a-5p, hsa-miR-28-3p, hsa-miR-151a-5p and hsa-miR-223-3p.

9. The reagent composition of claim 8, wherein, The primer is SEQ ID No.5-SEQ ID No.

13. SEQ ID No.5 is the reverse transcription primer of hsa-let-7a-5p, SEQ ID No.6 is the reverse transcription primer of hsa-miR-28-3p, SEQ ID No.7 is the reverse transcription primer of hsa-miR-151a-5p, SEQ ID No.8 is the reverse transcription primer of hsa-miR-223-3p, SEQ ID No.9 is the forward primer of hsa-let-7a-5p, SEQ ID No.10 is the forward primer of hsa-miR-28-3p, SEQ ID No.11 is the forward primer of hsa-miR-151a-5p, SEQ ID No.12 is the forward primer of hsa-miR-223-3p, and SEQ ID No.13 is the reverse primer of hsa-let-7a-5p, hsa-miR-28-3p, hsa-miR-151a-5p and hsa-miR-223-3p.

10. The reagent composition of claim 1, wherein, It also comprises DNA polymerase, dNTPs and ddH2O.

11. A diabetic retinopathy detection kit comprising the reagent composition of any one of claims 1-10.

12. The kit of claim 11, wherein Further comprising a positive quality control and a negative quality control.

13. The kit of claim 12, wherein The positive quality control is a synthetic cDNA fragment of hsa-let-7a-5p, hsa-miR-28-3p, hsa-miR-151a-5p and hsa-miR-223-3p.

14. The kit of claim 11, wherein Further comprising reagents for RNA extraction and / or reverse transcription.

15. The kit of claim 14, wherein The RNA extraction reagent is used for extracting RNA in a blood sample.

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