A primer-probe combination for detecting human TGFBI gene mutation and its application

Abstract

The present invention discloses a primer-probe composition for detecting human TGFBI gene mutations and its application, which belongs to the field of biotechnology. The technical problem to be solved by the present invention is how to quickly detect the R124H mutation of the human TGFBI gene. In order to solve the above technical problems, the present invention provides a primer-probe composition for detecting human TGFBI gene mutations, wherein the primer composition comprises a primer-probe composition 1 and a primer-probe composition 2, wherein the probe composition 1 comprises a primer pair and a probe for specifically amplifying and binding to a DNA molecule whose nucleotide sequence is SEQ ID No. 1, and the probe composition 2 comprises a primer pair and a probe for specifically amplifying and binding to a DNA molecule whose nucleotide sequence is SEQ ID No. 2. The present invention only requires one PCR amplification to obtain the result, is easy to operate, and eliminates the intermediate uncapping process, thereby reducing the risk of contamination.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a primer-probe combination for detecting human TGFBI gene mutation and an application thereof. Background Art

[0002] Corneal dystrophy is a general term for a group of hereditary, symmetrical, non-inflammatory corneal diseases characterized by the formation of deposits of varying shapes in the corneal tissue. Avellino corneal dystrophy (ACD) is an autosomal dominant disorder with high penetrance and roughly equal prevalence in males and females. Because its clinical manifestations are similar to those of granular corneal dystrophy (GCD) type II, namely, early onset (10-20 years of age), rapid progression, and rapid recurrence after surgery, this disorder was previously classified as GCD. However, in the late stages of the disease, linear opacities, or lattice patterns, may develop, particularly in the inferior cornea. Although this condition presents later, its prevalence increases significantly with age.

[0003] The TGFBI gene was the first gene discovered and is currently the most commonly reported gene associated with corneal dystrophy. It is located on human chromosome 5 and contains 17 exons, encoding a 683-amino acid protein. This protein is present in corneal epithelial and stromal cells, playing a role in wound healing and adhesion. Its pathology can lead to amyloid deposition. Avellino corneal dystrophy is primarily caused by a mutation at the p.R124H site (CGC > CAC) in exon 4 of the TGFBI gene. Clinical symptoms are cumulatively associated with the dose of the R124H mutation, meaning that patients with homozygous mutations have earlier and more severe onset of disease and are more likely to relapse than heterozygous patients.

[0004] The existing method for detecting R124H mutations uses quantitative fluorescence PCR plus pyrosequencing. This method requires real-time fluorescence PCR amplification followed by agarose gel electrophoresis. If a single target band appears, pyrosequencing is performed. Sequencing primers are added to the reaction and sequencing is performed on a QIAgen PyroMark Q24 sequencer. This requires three reactions per experiment, making the operation complex and requiring frequent opening of the sequencer, which increases the risk of aerosol contamination. Therefore, there is an urgent need to develop a simple, highly accurate method for detecting R124H mutations and related reagents or kits. Summary of the Invention

[0005] The technical problem to be solved by the present invention is: how to quickly detect whether the human TGFBI gene has an R124H mutation.

[0006] To solve the above technical problems, in the first aspect, the present invention provides a primer-probe composition for detecting human TGFBI gene mutations, wherein the primer-probe composition includes primer-probe composition 1 and primer-probe composition 2, wherein the primer-probe composition 1 includes a primer pair and a probe that specifically amplify and bind to a DNA molecule whose nucleotide sequence is SEQ ID No. 1, and the primer-probe composition 2 includes a primer pair and a probe that specifically amplify and bind to a DNA molecule whose nucleotide sequence is SEQ ID No. 2.

[0007] Furthermore, in the above primer-probe composition, the primer-probe composition 1 includes primer R124-F, primer R124wt-R and probe R124 probe, and the primer-probe composition 2 includes primer R124-F, primer R124H-R and probe R124 probe.

[0008] The primer R124-F is a single-stranded DNA whose nucleotide sequence is shown in SEQ ID No. 3;

[0009] The primer R124wt-R is a single-stranded DNA whose nucleotide sequence is shown in SEQ ID No. 4;

[0010] The primer R124H-R is a single-stranded DNA with a nucleotide sequence shown in SEQ ID No. 5;

[0011] The nucleotide sequence of the probe R124 probe is SEQ ID No.8.

[0012] In the present invention, the human TGFBI gene mutation may be the R124 mutation of the TGFBI gene, the R124 site is positions 22512-22514 of GenBank Accession No.NG_012646.1 (18-MAY-2020), and the HGVS with the R124H mutation is named NG_012646.1:g.22513G>A.

[0013] In the present invention, the DNA molecule with the nucleotide sequence of SEQ ID No. 1 is the wild type of human TGFBI gene R124, and the DNA molecule with the nucleotide sequence of SEQ ID No. 2 is the mutant type of human TGFBI gene R124H.

[0014] Furthermore, in the above-mentioned primer-probe composition, the primer-probe composition 1 and the primer-probe composition 2 further comprise a primer pair and a probe for an internal reference gene, the internal reference gene being the B2M gene, the primer pair for the internal reference gene comprising primer B2M-F and primer B2M-R, and the probe for the internal reference gene being B2M-probe,

[0015] The primer B2M-F is a single-stranded DNA whose nucleotide sequence is shown in SEQ ID No.6;

[0016] The primer B2M-R is a single-stranded DNA whose nucleotide sequence is shown in SEQ ID No.7;

[0017] The nucleotide sequence of the probe B2M-probe is SEQ ID No.9.

[0018] In the present invention, the R124-Probe is a probe that specifically recognizes the human TGFBI gene (including the wild-type R124 and the R124H mutant), and the B2M-Probe is a probe that specifically recognizes the B2M gene. The probe has a fluorescent group attached to its 5' end and a quencher group attached to its 3' end. The fluorescent groups attached to the 5' ends of the R124-Probe and the B2M-Probe have different luminescence types.

[0019] Fluorescent groups can be selected from, but not limited to, FAM (5 / 6-carboxyfluorescein), VIC (green fluorescent protein), TET (tetrachloro-6-carboxyfluorescein), JOE (2,7-dimethyl-4,5-dichloro-6-carboxyfluorescein), HEX (hexachloro-6-methylfluorescein), Cy3, TAMRA (6-carboxytetramethylrhodamine), ROX (carboxy-X-rhodamine), Texas Red, LC RED640, Cy5 (cyanine dye), LC RED705, FITC (fluorescein isothiocyanate), and other common fluorescent groups. The principle for selecting the fluorescent group of the probe in the primer composition for duplex PCR detection is that the color development of the two fluorescent groups is different.

[0020] The quenching group can be selected from at least one of TAMRA, BHQ1, BHQ2, BHQ3, MGB, and Dabcy1.

[0021] Furthermore, in the above-mentioned primer probe probe composition, the molar ratio of R124-F, R124wt-R, R124-probe, B2M-F, B2M-R, and B2M-probe in the primer probe composition 1 is 4:4:3:2:2:1; the molar ratio of R124-F, R124H-R, R124-probe, B2M-F, B2M-R, and B2M-probe in the primer probe composition 2 is 4:4:3:2:2:1.

[0022] Furthermore, in the above primer-probe combination, the primer-probe combination further comprises a quality control plasmid, and the quality control plasmid is selected from any one of the following:

[0023] A1), a recombinant plasmid containing a DNA molecule having a nucleotide sequence of SEQ ID No. 11;

[0024] A2), a recombinant plasmid containing a DNA molecule having a nucleotide sequence of SEQ ID No. 12;

[0025] A3), a recombinant plasmid containing a DNA molecule whose nucleotide sequence is SEQ ID No.13.

[0026] In one embodiment of the present invention, the above-mentioned quality control plasmids are divided into three groups, A, B, and C, for quality control in the operation process. The details are as follows:

[0027] Quality control product A contains two recombinant plasmids, A2) and A3);

[0028] Quality control product B contains three recombinant plasmids: A1), A2), and A3;

[0029] Quality control product C contains two recombinant plasmids, A1) and A3).

[0030] In order to solve the above technical problems, in a second aspect, the present invention provides the use of the above primer-probe combination in the preparation of a reagent or kit for identifying or assisting in identifying human TGFBI gene mutations.

[0031] To solve the above technical problems, in a third aspect, the present invention provides a reagent or kit for identifying or assisting in identifying human TGFBI gene mutations, wherein the reagent or kit contains the above primer-probe combination.

[0032] Furthermore, the above reagent or kit contains primer-probe composition 1 and primer-probe composition 2.

[0033] Furthermore, the molar ratio of R124-F, R124wt-R, R124-probe, B2M-F, B2M-R, and B2M-probe in the primer probe composition 1 in the above-mentioned reagent or kit is 4:4:3:2:2:1; the molar ratio of R124-F, R124H-R, R124-probe, B2M-F, B2M-R, and B2M-probe in the primer probe composition 2 is 4:4:3:2:2:1.

[0034] Furthermore, each component of the primer-probe combination in the above reagent or kit is packaged separately.

[0035] To solve the above technical problems, in a fourth aspect, the present invention provides a method for identifying or assisting in identifying the R124H mutation in the human TGFBI gene, comprising performing fluorescent quantitative PCR on the genomic DNA of a sample to be tested using the above-mentioned primer probe composition 1 and primer probe composition 2, respectively, obtaining the ΔCt of primer probe composition 1 and the ΔCt of primer probe composition 2 according to formula 1, and determining whether the sample to be tested has the mutation according to the following steps based on these two ΔCts:

[0036] R1) The ΔCt of primer-probe combination 1 is less than 5, and the ΔCt of primer-probe combination 2 is less than 5, indicating that the sample to be tested is a heterozygous mutation;

[0037] R2) The ΔCt of primer-probe combination 1 is ≥ 5, and the ΔCt of primer-probe combination 2 is < 5, indicating that the sample to be tested is a homozygous mutation;

[0038] R3) the ΔCt of primer-probe combination 1 is less than 5, and the ΔCt of primer-probe combination 2 is greater than or equal to 5, indicating that the sample to be tested is wild-type;

[0039] ΔCt=│Ct(R124-Probe)-Ct(B2M-Probe)│ Formula 1,

[0040] Wherein, Ct(R124-Probe) is the number of cycles of fluorescence quantitative PCR obtained with the probe R124-probe, and Ct(B2M-Probe) is the number of cycles of fluorescence quantitative PCR obtained with the probe B2M-probe. The number of cycles of fluorescence quantitative PCR obtained with the probe R124-probe represents the number of cycles of fluorescence quantitative PCR for the two reaction systems of primer-probe combination 1 and primer-probe combination 2, respectively.

[0041] In the embodiments of the present invention, Ct(R124-Probe) is also represented by Ct(FAM), and Ct(B2M-Probe) is also represented by Ct(ROX).

[0042] The present invention utilizes ARMS-PCR combined with fluorescent PCR probes. The principle is that base mismatches at the 3' end of the downstream PCR primers can lead to a dramatic reduction in product. Appropriately designed primers can directly distinguish mutant and wild-type genes through PCR. Prior art techniques design PCR primers specific to the SNP site of the gene being tested, combine them with Taqman probes for real-time fluorescent quantitative PCR, and simultaneously introduce an internal reference gene. The difference between the Ct values ​​of the test gene and the Ct reference gene (ΔCt) is used to determine the type of the polymorphic site, thereby determining the genotype of the corneal dystrophy gene mutation.

[0043] The present invention uses two reaction systems to detect the TGFBI gene R124H mutation site and R124wt wild-type site, according to Ct目标基因 With Ct 内参基因 The difference between the ΔCt values ​​is used to determine the type of the polymorphic site, thereby determining the genotype of the corneal dystrophy gene mutation. To reduce the risk of inaccurate interpretation results caused by inter-tube differences, an internal reference gene is introduced into each reaction system for simultaneous detection.

[0044] The above applications or methods are non-disease diagnosis applications or methods. The above applications or methods are not directly intended to obtain disease diagnosis results or health status of living human or animal bodies.

[0045] This patented detection method: 1) DNA is extracted from the sample and used as a DNA template. The DNA concentration is not less than 1ng / μL, and the OD260 / OD280 value of the DNA is between 1.6-2.0; 2) The amplification reaction system of this kit is: 25×Anstart MasterPCR Mix 0.8μL, 5×Taq Buffer (MgCl2) 2+ 3) PCR reaction system: 50°C for 2 min, 95°C for 3 min; 95°C for 30 sec, 60°C for 50 sec, 40 cycles; 25°C for 1 min; 4) Interpretation of results.

[0046] Interpretation of results:

[0047] R124 interpretation results: The amplification curve of the B2M gene fluorescence PCR showed a typical S-shape, with a Ct value ≤ 30, ΔCt = |Ct(R124-Probe)-Ct(B2M-Probe)|.

[0048] (2) Technical effects

[0049] The present invention only requires one PCR amplification (the two groups are performed in the same reaction program and on the same reaction plate, which can be considered as the same PCR amplification reaction) to obtain the results. The operation is simple and the intermediate opening process is omitted, thereby reducing the risk of contamination. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 The primer-probe combination judgment results and first-generation sequencing results of sample 1 in accuracy verification are shown.

[0051] Figure 2 The primer-probe combination judgment results and first-generation sequencing results of sample 2 in accuracy verification are shown.

[0052] Figure 3 The primer-probe combination judgment results and first-generation sequencing results of sample 3 in accuracy verification are shown.

[0053] Figure 4The primer-probe combination judgment results and first-generation sequencing results of sample 4 in accuracy verification are shown.

[0054] Figure 5 The primer-probe combination judgment results and first-generation sequencing results of sample 5 in accuracy verification are shown.

[0055] Figure 6 This is the test result of sample-1 at 10 ng / μL in the detection line determination experiment.

[0056] Figure 7 This is the test result of sample-1 at 1 ng / μL in the detection line determination experiment.

[0057] Figure 8 This is the test result of sample-1 at 100 pg / μL in the detection line determination experiment.

[0058] Figure 9 This is the test result of sample-2 at 10 ng / μL in the detection line determination experiment.

[0059] Figure 10 This is the test result of sample-2 at 1 ng / μL in the detection line determination experiment.

[0060] Figure 11 This is the test result of sample-2 at 100 pg / μL in the detection line determination experiment.

[0061] Figure 12 This is the test result of sample-1 at 2 ng / μL in the test line verification experiment.

[0062] Figure 13 This is the test result of sample-2 at 2 ng / μL in the test line verification experiment.

[0063] Figure 14 This is the test result of sample-1 at 1 ng / μL in the test line verification experiment.

[0064] Figure 15 This is the test result of sample-2 at 1 ng / μL in the test line verification experiment.

[0065] Figure 16 This is the test result of sample-1 at 500 pg / μL in the test line verification experiment.

[0066] Figure 17 This is the test result of sample-2 at 500 pg / μL in the test line verification experiment. DETAILED DESCRIPTION

[0067] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0068] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0069] Example 1: Design of primers for detecting R124H site

[0070] 1.1 Nucleotide sequence of the R124H site

[0071] The present invention uses ARMS-PCR combined with a fluorescent PCR probe method to detect the p.R124H mutation (CGC>CAC) in the fourth exon of the TGFBI gene. Primers are designed near the p.R124H mutation (CGC>CAC) in the fourth exon of the TGFBI gene to detect the presence of the R124H mutation. Using the B2M gene as an internal reference, the primers are designed as shown in Table 1:

[0072] Table 1: Primer sequences

[0073]

[0074] 1.2. Primer-probe combination

[0075] The primer-probe combinations are divided into two groups according to the monitoring purpose, and the specific groups are as follows:

[0076] Primer-probe combination 1: R124-F, R124wt-R, R124 probe, B2M-F, B2M-R, and B2M-probe;

[0077] Primer-probe combination 2: R124-F, R124H-R, R124 probe, B2M-F, B2M-R, and B2M-probe;

[0078] Primer-probe combination 1 was used to amplify the wild-type R124 DNA fragment shown in SEQ ID No. 1 and the internal reference B2M; primer-probe combination 2 was used to amplify the R124H mutant DNA fragment shown in SEQ ID No. 2 and the internal reference B2M. The amounts of each component in the two primer-probe combinations are shown in the table below. TE was used to prepare the mixture to the final concentrations shown in the table below.

[0079] Table 2: Final concentration of primer combination in the reaction system

[0080]

[0081] The above detection method also introduces quality control products to operate simultaneously with the samples to monitor the entire operation process and increase the credibility of the experimental results.

[0082] Construction of quality control plasmid PUC-SP-R124H: Use the DNA molecule with the nucleotide sequence of SEQ ID No.11 to insert the fragment between the EcoRV enzyme recognition sites of the vector PUC-SP, keep the other nucleotide sequences of the vector PUC-SP unchanged, and sequence verification to obtain the recombinant plasmid PUC-SP-R124H correctly.

[0083] Construction of quality control plasmid PUC-SP-R124WT: Use the DNA molecule with the nucleotide sequence of SEQ ID No.12 to insert the fragment between the EcoRV enzyme recognition sites of the vector PUC-SP, keep the other nucleotide sequences of the vector PUC-SP unchanged, and sequence verification to obtain the recombinant plasmid PUC-SP-R124WT correctly.

[0084] Construction of quality control plasmid PUC-SP-B2M: Use the DNA molecule with the nucleotide sequence of SEQ ID No.13 to insert the fragment between the SmaI enzyme recognition sites of the vector PUC-SP, keep the other nucleotide sequences of the vector PUC-SP unchanged, and sequence to verify the correct acquisition of the recombinant plasmid PUC-SP-B2M.

[0085] Among them, the nucleotide sequence of the complete sequence of vector PUC-SP is SEQ ID No.10.

[0086] Quality control product A contains quality control plasmids PUC-SP-R124WT and PUC-SP-B2M;

[0087] Quality control product B contains quality control plasmids PUC-SP-R124WT, PUC-SP-R124H, and PUC-SP-B2M;

[0088] Control C contains the control plasmids PUC-SP-R124H and PUC-SP-B2M.

[0089] 1.3 Detection Method

[0090] The specific detection method is as follows:

[0091] 1) Extract DNA from the sample and use it as a DNA template. The DNA concentration should be no less than 1 ng / μL. The OD value of the DNA should be 260 / OD 280 The value is between 1.6-2.0, and the OD260 / OD230 value is between 1.6-2.0;

[0092] 2) Reaction system

[0093] Reaction system A: 25×Anstart Master PCR Mix 0.8μL, 5×Taq Buffer (Mg 2+ Plus) 4 μL, 2 μL of 10× primer-probe combination 1 mixture, 3 μL of template, and ultrapure water to 20 μL;

[0094] Reaction system B: 25×Anstart Master PCR Mix 0.8μL, 5×Taq Buffer (Mg 2+ Plus) 4 μL, 2 μL of 10× primer-probe combination 2, 3 μL of template, and ultrapure water to 20 μL;

[0095] 3) PCR reaction system: 50°C for 2 min, 95°C for 3 min; 95°C for 30 sec, 60°C for 50 sec, 40 cycles; 25°C for 1 min;

[0096] 4) Interpretation of results

[0097] R124 interpretation results: The amplification curve of the B2M gene fluorescence PCR showed a typical S-shaped curve, and the Ct value was ≤30, ΔCt=│Ct(R124-Probe)-Ct(B2M-Probe)│Equation 1,

[0098] Wherein, Ct(R124-Probe) is the number of cycles experienced when the FAM signal reaches the set threshold (exponential amplification), and Ct(B2M-Probe) is the number of cycles experienced when the ROX signal reaches the set threshold (exponential amplification).

[0099] ΔCt R124H (Reaction system B) and ΔCt R124wt (Reaction system A) represents the absolute value of the difference between Ct(R124-Probe) and Ct(B2M-Probe) in the two reaction systems.

[0100] Interpret the results according to Table 3.

[0101] Table 3: Criteria for interpretation of results

[0102]

[0103] Example 2: Verification of detection accuracy

[0104] Representative clinical samples of 1-5 people were selected and tested according to the detection method shown in 1.3 of Example 1. The results are shown in the following table and Figure 1-5 As shown, Figure 1-5 The left side of the center shows the fluorescence detection results of this patented kit, and the right side shows the results of 124-site first-generation sequencing. The results show that the results of the primer combination provided by this application for samples 1-5 are consistent with the first-generation sequencing results. The clinical samples are DNA extracted from blood samples obtained from a partner hospital, and the patient has signed an informed consent form.

[0105] Table 4: Interpretation results of samples 1-5

[0106]

[0107] Example 3: Sensitivity Detection

[0108] 3.1. Detection limit determination

[0109] One R124 wild-type and one R124H mutant clinical sample (sample 1 and sample 3 in Example 2) were selected and diluted to three concentration gradients of 10 ng / μL, 1 ng / μL, and 100 pg / μL, respectively. Each concentration gradient was repeated 4 times to find the minimum detection limit while ensuring the consistency of positive and negative results.

[0110] The threshold for interpretation was set to 30,000, and the baseline was automatically set. The sample internal reference required the amplification curve to be a typical S-shape and the Ct value to be ≤30. The mutation status of the sample was determined by calculating the difference between the Ct values ​​of the FAM channel and the ROX channel in each reaction tube of the test sample.

[0111] ΔCt=│Ct(R124-Probe)-Ct(B2M-Probe)│ Equation 1

[0112] When the ΔCt of reaction system B is less than 5, regardless of the result of reaction system A, it is judged as R124H mutation; when the ΔCt of reaction system B is ≥ 5, and the ΔCt of reaction system A is less than 5, it is judged as a sample without R124H mutation.

[0113] Table 1 shows that when the sample concentration is 100 pg / μL, CtROX>30, which does not meet the interpretation standard; when the sample concentration is 1 ng / μL, CtROX is close to 30, and the interpretation result is consistent with the known genotype of the sample. Therefore, 1 ng / μL is initially determined as the detection limit concentration of this patented kit.

[0114] The data statistics are shown in Table 5: (wherein, sample-1 is a heterozygous type (sample 1 in the embodiment), and sample-2 is a wild type (sample 3 in the embodiment 1))

[0115] Table 5: Detection limit determination data statistics

[0116]

[0117]

[0118] 3.2 Detection limit verification

[0119] The above two clinical samples were diluted to three concentration gradients of 2ng / μL, 1ng / μL and 500pg / μL, respectively. Each concentration gradient was repeated 20 times for verification of the minimum detection limit.

[0120] When the detection concentration was 1 ng / μL, one of the 20 repetitions in the sample-1B system had a detection result that did not match the actual sample type, with a detection compliance rate of 95% (data shown in Table 5). When the detection concentration was 500 pg / μL, five of the 20 repetitions in the sample-1B system had a detection result that did not match the actual sample type, with a detection compliance rate of 75% (data shown in Table 6). Therefore, 1 ng / μL was determined to be the minimum detection limit of this patented kit (referring to the "Technical Review Guidelines for Registration of Gene Mutation Detection Reagents Related to Hereditary Deafness," which stipulates that the detection limit requirement is to use a concentration with a 95% detection rate as the minimum detection limit). The detection rates of both the Sample-1A and Sample-2A systems were 100%.

[0121] Table 6: Detection limit verification data statistics

[0122]

[0123]

[0124] Note: “ / ” in the table means Undetermined, i.e., the Ct value was not detected.

[0125] Table 7:

[0126]

[0127]

[0128]

[0129] Note: “ / ” in the table means Undetermined, i.e., the Ct value was not detected.

[0130] Table 8:

[0131]

[0132]

[0133] Note: “ / ” in the table means Undetermined, i.e., the Ct value was not detected.

[0134] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. A primer-probe combination for detecting human TGFBI gene mutations, characterized in that: The primer-probe composition includes primer-probe composition 1 and primer-probe composition 2, wherein the primer-probe composition 1 includes a primer pair and a probe that specifically amplify and bind to a DNA molecule whose nucleotide sequence is SEQ ID No. 1, and the primer-probe composition 2 includes a primer pair and a probe that specifically amplify and bind to a DNA molecule whose nucleotide sequence is SEQ ID No. 2; The primer-probe composition 1 includes primer R124-F, primer R124wt-R and probe R124-probe, and the primer-probe composition 2 includes primer R124-F, primer R124H-R and probe R124-probe. The primer R124-F is a single-stranded DNA whose nucleotide sequence is shown in SEQ ID No. 3; The primer R124wt-R is a single-stranded DNA whose nucleotide sequence is shown in SEQ ID No. 4; The primer R124H-R is a single-stranded DNA with a nucleotide sequence shown in SEQ ID No. 5; The nucleotide sequence of the probe R124-probe is SEQ ID No.

8.

2. The primer-probe combination according to claim 1, wherein: The primer-probe composition 1 and the primer-probe composition 2 further comprise a primer pair and a probe for an internal reference gene, wherein the internal reference gene is the B2M gene, the primer pair for the internal reference gene comprises primer B2M-F and primer B2M-R, the probe for the internal reference gene is B2M-probe, and the primer B2M-F is a single-stranded DNA having a nucleotide sequence shown in SEQ ID No. 6; The primer B2M-R is a single-stranded DNA whose nucleotide sequence is shown in SEQ ID No.7; The nucleotide sequence of the probe B2M-probe is SEQ ID No.

9.

3. The primer-probe combination according to claim 2, wherein: The molar ratio of R124-F, R124wt-R, R124-probe, B2M-F, B2M-R, and B2M-probe in the primer probe composition 1 is 4:4:3:2:2:1; the molar ratio of R124-F, R124H-R, R124-probe, B2M-F, B2M-R, and B2M-probe in the primer probe composition 2 is 4:4:3:2:2:

1.

4. The primer-probe combination according to claim 2, wherein: The primer-probe combination further comprises a quality control plasmid, and the quality control plasmid is selected from any one of the following: A1), a recombinant plasmid containing a DNA molecule having a nucleotide sequence of SEQ ID No. 11; A2), a recombinant plasmid containing a DNA molecule having a nucleotide sequence of SEQ ID No. 12; A3), a recombinant plasmid containing a DNA molecule whose nucleotide sequence is SEQ ID No.

13.

5. Use of the primer-probe combination according to any one of claims 1 to 4 in the preparation of a reagent or kit for identifying or assisting in identifying a human TGFBI gene mutation.

6. A reagent or kit for identifying or assisting in identifying the R124H mutation in the human TGFBI gene, characterized in that: The reagent or kit contains the primer-probe combination according to any one of claims 1 to 4.

7. The reagent or kit according to claim 6, characterized in that: The reagent or kit contains a primer-probe composition 1 and a primer-probe composition 2.

8. The reagent or kit according to claim 6, characterized in that: The molar ratio of R124-F, R124wt-R, R124-probe, B2M-F, B2M-R, and B2M-probe in the primer-probe composition 1 in the reagent or kit is 4:4:3:2:2:1; the molar ratio of R124-F, R124H-R, R124-probe, B2M-F, B2M-R, and B2M-probe in the primer-probe composition 2 is 4:4:3:2:2:1.

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