Genetic test reagents, kits for corneal dystrophy

Abstract

The application provides a gene detection kit for corneal dystrophy, comprising blood sample pretreatment reagents and amplification reagents, wherein the blood sample pretreatment reagents comprise 1-50 mM Tris, 1-20 mM isothiocyanate guanidine, 20-80 mM lauryl alcohol, 300-500 mM erythritol, 5000-8000 U / mL neutral protease, and the rest is water, and the pH is 6.5-7.5; and the amplification reagents comprise amplification primers and probes. When the kit is used for detecting corneal dystrophy from a whole blood sample, the operation is simple, the time consumption is short, no additional nucleic acid extraction operation is needed, the detection accuracy of the kit is high, the kit can be used for etiological diagnosis and gene screening of corneal dystrophy, and the clinical application prospect is wide.

Description

Technical Field

[0001] This invention belongs to the field of gene detection technology, specifically relating to gene detection reagents and kits for corneal dystrophy. Background Technology

[0002] Corneal dystrophy is a collective term for a series of primary, progressive corneal diseases with a familial genetic component. Most cases are autosomal dominant; they originate in the cornea, primarily affecting the central cornea, and are symmetrical in both eyes; the disease progresses slowly, and neovascularization is often absent in the affected area; initially, only a single layer of the cornea is affected; in later stages, it can involve adjacent layers and even affect the entire cornea; drug treatment is ineffective. Corneal transplantation surgery can be performed to treat those whose vision is impaired.

[0003] Early genetic screening for corneal dystrophy is beneficial for early intervention and preventing the condition from worsening later. Current technologies have disclosed mutation sites in several genes associated with corneal dystrophy, such as the TGFβI, CHST6, BIGH3, and TMCO3 genes. Diagnosis of corneal dystrophy primarily involves designing primers and probes targeting these mutation sites, extracting DNA for PCR amplification, and sequencing the DNA.

[0004] Chinese patent CN201711147057.4 discloses a primer probe for diagnosing corneal dystrophy caused by mutations at the 124 site of the human TGFβI gene. The primer includes specific primers designed for the 124 site of the TGFβI gene: a specific wild-type Taqman fluorescent probe targeting the wild-type site R124, and at least one of four specific mutant Taqman fluorescent probes targeting mutant sites C124, S124, H124, and L124. Each probe is linked to a different fluorescent reporter group, allowing for separate monitoring by collecting different fluorescence signals, without the need for an internal control. A detection method for human TGFβI gene mutations at the 124 site based on the above primer probes is also provided, which has advantages such as high sensitivity, strong specificity, low contamination, good accuracy, and convenience, making it suitable for application in clinical case analysis and laboratory work.

[0005] Chinese patent CN201611138588.2 discloses a kit for detecting mutation sites of susceptibility genes for granular keratopathy. The kit of this invention includes primer pair A, primer pair B, primer pair C, and extension primers A1, A2, B, and C; this kit can detect nucleotides of rs121909208, rs121909209, rs121909211, and rs121909215, and can also be used to assist in the diagnosis of granular keratopathy.

[0006] However, current technologies for the genetic diagnosis of corneal dystrophy require DNA extraction followed by amplification, which is a complex and time-consuming process. Developing a more efficient test kit is essential for clinical use. Summary of the Invention

[0007] To address the aforementioned issues, this invention aims to provide a gene detection kit that can directly use blood samples for corneal dystrophy, simultaneously amplifying multiple relevant loci, with short processing time and easy operation, enabling faster and more accurate detection.

[0008] On the one hand, the present invention provides a gene detection kit for corneal dystrophy.

[0009] Specifically, the kit includes blood sample pretreatment reagents and amplification reagents.

[0010] More specifically, the blood sample pretreatment reagent includes: 1-50mM Tris, 1-20mM guanidine isosulfate, 20-80mM lauryl alcohol, 300-500mM erythritol, 5000-8000U / mL neutral protease, with the remainder being water, and a pH of 6.5-7.5; the amplification reagent includes amplification primers and probes.

[0011] Preferably, the pretreatment reagent includes: 20-40mM Tris, 10-20mM guanidine isosulfate, 20-60mM lauryl alcohol, 400-500mM erythritol, and 5000-6000U / mL neutral protease.

[0012] More preferably, the pretreatment reagent includes: 25-40mM Tris, 10-15mM guanidine isosulfate, 50-60mM lauryl alcohol, 400-500mM erythritol, and 5000-6000U / mL neutral protease.

[0013] Furthermore, the pretreatment reagents include: 25mM Tris, 10mM guanidine isosulfate, 50mM lauryl alcohol, 500mM erythritol, and 5000U / mL neutral protease.

[0014] The kit described can be used directly for blood sample amplification detection. The blood sample can be plasma or serum.

[0015] Specifically, the amplification primers and probes are used to detect TGFβI gene mutations: c.370C>T (rs121909210), c.371G>T / A (rs121909211), c.1663C>T (rs121909208), and c.1664G>A (rs121909209).

[0016] Preferably, the amplification primers and probes are as follows:

[0017]

[0018] The probe sequence is labeled as follows:

[0019] SEQ ID NO.5: The 5' end is marked with VIC, and the 3' end is marked with MGB;

[0020] SEQ ID NO.6: The 5' end is labeled FAM, and the 3' end is labeled MGB;

[0021] SEQ ID NO.7: The 5' end is marked with VIC, and the 3' end is marked with MGB;

[0022] SEQ ID NO.8: The 5' end is labeled FAM, and the 3' end is labeled MGB;

[0023] SEQ ID NO.9: ROX is marked at the 5' end, and MGB is marked at the 3' end;

[0024] SEQ ID NO.10: The 5' end is marked with VIC, and the 3' end is marked with MGB;

[0025] SEQ ID NO.11: The 5' end is labeled FAM, and the 3' end is labeled MGB;

[0026] SEQ ID NO.12: The 5' end is labeled FAM, and the 3' end is labeled MGB.

[0027] The concentration of primers or probes used is 0.5-1.5 μM.

[0028] The kit also includes a PCR amplification buffer system, which comprises: 100mM Tris-HCl, 10-15mM MgCl2, 20-30mM KCl, and 20-50mM glacial acetic acid.

[0029] Preferably, the buffer system comprises: 100mM Tris-HCl, 12-14mM MgCl2, 20-25mM KCl, and 20-30mM glacial acetic acid.

[0030] More preferably, the buffer system comprises: 100mM Tris-HCl, 14mM MgCl2, 22mM KCl, and 25mM glacial acetic acid.

[0031] The kit may also include other reagents for PCR amplification.

[0032] Preferably, the amplification system suitable for the kit is as follows:

[0033] PCR amplification buffer system (10×) 2.5 DNA polymerase (5 U / μL) 1 dNTPs (2.5mM each) 2 Primer and probe mixture 3(3.5) template 5 <![CDATA[ddH2O]]> Make up to 25

[0034] The amplification program applicable to the kit is: 95℃ for 5 min, (95℃ for 10 s, 60℃ for 30 s fluorescence acquisition) for 40 cycles.

[0035] The kit may also include positive control and negative control.

[0036] On the other hand, the present invention provides a blood sample pretreatment reagent.

[0037] Specifically, the blood sample pretreatment reagent includes: 1-50mM Tris, 1-20mM guanidine isosulfate, 20-80mM lauryl alcohol, 300-500mM erythritol, 5000-8000U / mL neutral protease, with the remainder being water, and a pH of 6.5-7.5.

[0038] The blood sample pretreatment reagent is added directly to the blood sample for use.

[0039] Preferably, the blood sample pretreatment reagent includes: 20-40mM Tris, 10-20mM guanidine isosulfate, 20-60mM lauryl alcohol, 400-500mM erythritol, and 5000-6000U / mL neutral protease.

[0040] More preferably, the pretreatment reagent includes: 25-40mM Tris, 10-15mM guanidine isosulfate, 50-60mM lauryl alcohol, 400-500mM erythritol, and 5000-6000U / mL neutral protease.

[0041] Furthermore, the pretreatment reagents include: 25mM Tris, 10mM guanidine isosulfate, 50mM lauryl alcohol, 500mM erythritol, and 5000U / mL neutral protease.

[0042] In another aspect, the present invention provides a mixture of amplification primers and probes for detecting corneal dystrophy.

[0043] The mixture was used to amplify TGFβI gene mutations: c.370C>T (rs121909210), c.371G>T / A (rs121909211), c.1663C>T (rs121909208), and c.1664G>A (rs121909209).

[0044] This invention also provides the application of the aforementioned blood sample pretreatment reagent and primer-probe mixture in the preparation of a kit for detecting corneal dystrophy.

[0045] The kit described is a blood sample testing kit.

[0046] The kit may also include other reagents for PCR amplification.

[0047] Other reagents used for PCR amplification include, but are not limited to: DNA polymerase, positive control, negative control, dNTPs, etc.

[0048] The beneficial effects of this invention are:

[0049] The kit provided by this invention is simple to operate and quick to use when detecting corneal dystrophy from whole blood samples. It does not require additional nucleic acid extraction and has high detection accuracy. It can be used for the diagnosis of corneal dystrophy and gene screening, and has broad clinical application prospects. Detailed Implementation

[0050] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0051] In the following embodiments:

[0052] All enzymes used for PCR amplification were Taq DNA polymerase, purchased from Takara (product number R001A) at a concentration of 5 U / μL.

[0053] The 10×Buffer was purchased from Takara, item number R001A.

[0054] Positive control samples consist of recombinant plasmids that are positive for each detection target, and the solvent is human serum;

[0055] The negative control was human serum;

[0056] The human serum was purchased from Merck, catalog number SAE0012.

[0057] The neutral protease was purchased from Solarbio, catalog number Z8031.

[0058] Example 1

[0059] The kit in this embodiment includes the following components:

[0060] (1) Blood sample pretreatment reagents: 25mM Tris, 10mM guanidine isosulfate, 50mM lauryl alcohol, 500mM erythritol, 5000U / mL neutral protease, and the remainder is water; pH is 7.

[0061] (2) PCR amplification buffer: 100mM Tris-HCl, 15mM MgCl2, 20mM KCl, 30mM glacial acetic acid.

[0062] (3) DNA polymerase: 5 U / μL.

[0063] (4) dNTPs: 2.5mM each.

[0064] (5) Primer-probe mixture

[0065]

[0066] The probe sequence is labeled as follows:

[0067] SEQ ID NO.5: The 5' end is marked with VIC, and the 3' end is marked with MGB;

[0068] SEQ ID NO.6: The 5' end is labeled FAM, and the 3' end is labeled MGB;

[0069] SEQ ID NO.7: The 5' end is marked with VIC, and the 3' end is marked with MGB;

[0070] SEQ ID NO.8: The 5' end is labeled FAM, and the 3' end is labeled MGB;

[0071] SEQ ID NO.9: ROX is marked at the 5' end, and MGB is marked at the 3' end;

[0072] SEQ ID NO.10: The 5' end is marked with VIC, and the 3' end is marked with MGB;

[0073] SEQ ID NO.11: The 5' end is labeled FAM, and the 3' end is labeled MGB;

[0074] SEQ ID NO.12: The 5' end is labeled FAM, and the 3' end is labeled MGB.

[0075] Each primer was at a concentration of 10 μM, and each probe was at a concentration of 10 μM.

[0076] Example 2

[0077] The difference from Example 1 is that the blood sample pretreatment reagents are: 40mM Tris, 15mM guanidine isosulfate, 60mM lauryl alcohol, 400mM erythritol, 6000U / mL neutral protease, and the remainder is water; the pH is 7.

[0078] Example 3

[0079] The difference from Example 1 is that the blood sample pretreatment reagents are: 20mM Tris, 20mM guanidine isosulfate, 50mM lauryl alcohol, 400mM erythritol, 5000U / mL neutral protease, and the remainder is water; the pH is 7.

[0080] Example 4

[0081] The difference from Example 1 is that the PCR amplification buffer system includes: 100mM Tris-HCl, 10mM MgCl2, 30mM KCl, and 40mM glacial acetic acid.

[0082] Example 5: A method for detecting corneal dystrophy

[0083] Taking the kit from Example 1 as an example, the specific operating steps for detecting malnutrition according to the present invention are explained as follows:

[0084] (1) Collect blood samples, let them stand for 5-10 minutes, then add blood sample pretreatment reagent to the blood samples. The volume ratio of blood sample to pretreatment reagent is 2:1. Mix gently and let stand for 5 minutes before using as a template for PCR amplification.

[0085] (2) The PCR amplification system is as follows:

[0086]

[0087]

[0088] The reaction was divided into 4 wells for qPCR detection.

[0089] (3) The PCR amplification program is: 95℃ for 5 min, (95℃ for 10 s, 60℃ for 30 s fluorescence collection) for 40 cycles.

[0090] (4) Result determination method:

[0091] Genotype determination can only be performed when the yin-yang control sample meets all three of the following conditions.

[0092] Negative controls: The amplification curves of each detection target have no exponential growth phase or the Ct value is >35;

[0093] Positive control samples: The amplification curves of each detection target show an exponential growth phase and the Ct value is ≤35;

[0094] Both of the above conditions must be met simultaneously in the same test; otherwise, the test will be considered invalid and must be repeated.

[0095]

[0096] Experiment Example 1: Population Sample Detection

[0097] The efficacy of this invention was verified by selecting 20 patients with corneal dystrophy and 20 healthy individuals.

[0098] The clinical diagnostic results for each patient are as follows:

[0099] 1 Reis-Buckler's corneal dystrophy 11 Fuchs corneal endothelial dystrophy 2 Avellino corneal dystrophy 12 Avellino corneal dystrophy 3 Avellino corneal dystrophy 13 Lattice corneal dystrophy type I 4 Fuchs corneal endothelial dystrophy 14 Avellino corneal dystrophy 5 Corneal epithelial and Bowman's layer dystrophy 15 Thiel-Behnke corneal dystrophy 6 plaque corneal dystrophy 16 Fuchs corneal endothelial dystrophy 7 Avellino corneal dystrophy 17 Gel-droplet corneal dystrophy 8 Granular corneal dystrophy type I 18 Granular corneal dystrophy type I 9 Avellino corneal dystrophy 19 Avellino corneal dystrophy 10 Granular corneal dystrophy type I 20 Avellino corneal dystrophy

[0100] After blood was collected, it was tested using the kits described in Examples 1-4. Five patients tested positive (numbered 2, 9, 13, 14, and 18), while all healthy individuals tested negative.

[0101] To confirm the effectiveness of the kit, relevant genes in patient samples were tested. The results showed that no target mutation was found in the samples that did not test positive, indicating that the kit of the present invention is effective at least for the diagnosis of corneal dystrophy caused by the target mutation.

[0102] Example 2: Anti-interference capability of the reagent kit

[0103] Direct PCR testing using blood samples can be affected by the components in the blood sample, especially the anticoagulant added after sampling. Therefore, it is necessary to test the anti-interference ability of the test kit.

[0104] Considering the issue of blood volume, the five positive samples from Example 1 were used to verify the anti-interference ability against different concentrations of anticoagulants. Two anticoagulants were tested: EDTA and heparin. The dosage of EDTA was set at 0.5 g / L, 1 g / L, and 1.5 g / L; the dosage of heparin was set at 10 IU / mL and 12 IU / mL.

[0105] The specific settings are as follows:

[0106]

[0107]

[0108] The fact that positive results could still be detected in each of the above groups under conditions containing different anticoagulants indicates that the kit of the present invention has good anti-interference ability, has anti-interference ability against at least 1.5 g / L EDTA, and has anti-interference ability against at least 12 IU / mL heparin.

[0109] The anti-interference ability of the kits in Examples 1-4 was tested using positive control samples. The concentrations of EDTA were set at 0.5 g / L, 1 g / L, 1.5 g / L, and 2 g / L; the concentrations of heparin were set at 10 IU / mL, 12 IU / mL, and 14 IU / mL. The results of the anti-interference concentrations of EDTA and heparin are as follows:

[0110] Example 1 2g / L 14 IU / mL Example 2 2g / L 14 IU / mL Example 3 2g / L 14 IU / mL Example 4 2g / L 14 IU / mL

[0111] Experimental Example 3: Reagent Kit Sensitivity

[0112] The sensitivity of the reagent kit was tested using quality control materials.

[0113] Quality control samples of different concentrations were tested. The concentrations of the quality control samples were set at 0.1 ng / μL, 0.5 ng / μL, 1 ng / μL and 2 ng / μL respectively. The results showed that the limit of detection of the kits in Examples 1-4 of this application was 0.1 ng / μL.

[0114] Comparative Example

[0115] The comparative example is set up with reference to Example 1, as follows:

[0116]

[0117]

[0118] Following the method described in Experiment 1, five positive samples were tested, and the results are as follows:

[0119] Comparative Example 1: Positive mutations could still be detected in 5 samples, but the Ct values ​​of the VIC and FAM channels increased by 1-3.

[0120] Comparative Example 2: 3 out of 5 samples failed to detect positive mutations.

[0121] Comparative Example 3: Two out of five samples failed to detect positive mutations.

[0122] Comparative Example 4: One out of five samples failed to detect a positive mutation.

[0123] Following the method in Experimental Example 2, the anti-interference ability of Comparative Examples 1-4 for samples 2, 9, 13, and 14 was tested, and the results are as follows:

[0124] Comparative Example 1 (Sample 2) 1g / L 10 IU / mL Comparative Example 2 (9 samples tested) <0.5g / L (no results found within the set range) <10 IU / mL Comparative Example 3 (Sample 13) 1g / L 10 IU / mL Comparative Example 4 (Sample 14) 0.5g / L 10 IU / mL

[0125] To determine if the issue was due to sample quality, sample 18 was tested using the same kit as Comparative Example 2 to assess its anti-interference capability. The results showed that within the set ranges for EDTA and heparin, Comparative Example 2 still failed to detect any results, indicating that its anti-interference concentration against EDTA was <0.5 g / L and against heparin was <10 IU / mL.

[0126] The anti-interference ability of the kits in Comparative Examples 1-4 was tested, and the results are as follows:

[0127] Comparative Example 1 0.5g / L 12 IU / mL Comparative Example 2 2g / L 14 IU / mL Comparative Example 3 2g / L 14 IU / mL Comparative Example 4 2g / L 14 IU / mL

Claims

1. A gene detection kit for corneal dystrophy, comprising blood sample pretreatment reagents and amplification reagents, characterized in that, The blood sample pretreatment reagent consists of: 25mM Tris, 10mM guanidine isothiocyanate, 50mM lauryl alcohol, 500mM erythritol, 5000U / mL neutral proteinase, with the remainder being water, and a pH of 7.0; the amplification reagent includes amplification primers and probes. It also includes a PCR amplification buffer system, which consists of: 100mM Tris-HCl, 15mM MgCl2, 20mM KCl, and 30mM glacial acetic acid.

2. The reagent kit according to claim 1, characterized in that, It can be used directly for blood sample amplification detection.

3. The reagent kit according to claim 1, characterized in that, The amplification primers and probes described herein are used to detect TGFβI gene mutations: rs121909210 c.370C>T, rs121909211 c.371G>T / A, rs121909208 c.1663C>T, and rs121909209 c.1664G>A.

4. The reagent kit according to claim 3, characterized in that, The amplification primers are SEQ ID NO.1-4, and the probes are SEQ ID NO.5-12.

5. The reagent kit according to claim 4, characterized in that, It also includes DNA polymerase, dNTPs, and quality control products.

6. The reagent kit according to claim 5, characterized in that, It also includes positive control samples and negative control samples.

7. A blood sample pretreatment reagent for PCR amplification, characterized in that, The composition is: 25mM Tris, 10mM guanidine isothiocyanate, 50mM lauryl alcohol, 500mM erythritol, 5000U / mL neutral protease, with the balance being water, and the pH is 7.

0. The PCR amplification buffer system used for PCR detection is: 100mM Tris-HCl, 15mM MgCl2, 20mM KCl, and 30mM glacial acetic acid.

8. A mixture of amplification primers and probes for detecting corneal dystrophy, characterized in that, The amplification primers are SEQ ID NO.1-4, and the probes are SEQ ID NO.5-12; The blood sample pretreatment reagent and PCR amplification buffer system are used for amplification. The blood sample pretreatment reagent consisted of: 25 mM Tris, 10 mM guanidine isothiocyanate, 50 mM lauryl alcohol, 500 mM erythritol, 5000 U / mL neutral protease, with the remainder being water, and a pH of 7.

0. The PCR amplification buffer system consisted of 100 mM Tris-HCl, 15 mM MgCl2, 20 mM KCl, and 30 mM glacial acetic acid.

9. The use of the mixture of claim 8 in the preparation of a kit for detecting corneal dystrophy.

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