Primer group, kit and method for detecting Fabry cardiomyopathy genetic susceptibility gene
By designing specific primer sets and kits, and combining PCR amplification and Sanger sequencing, the accuracy problem of gene detection for Fabry cardiomyopathy was solved, and efficient and low-cost detection of GLA gene c.640-801G>A site mutation was achieved, which is suitable for early screening in primary healthcare institutions.
Patent Information
- Application Number
- CN202511628434.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-07
- Publication Date
- 2026-01-27
AI Technical Summary
Existing technologies for gene testing of Fabry cardiomyopathy suffer from complex data interpretation, uneven coverage, inability to reliably detect certain mutations, and limited ability to detect repetitive sequences, leading to false negative results and an inability to accurately identify GLA gene c.640-801G>A site mutations.
We designed a specific primer set and kit, including PCR amplification primers and Sanger sequencing primers. Through PCR amplification, enzyme purification and Sanger sequencing reaction, we accurately detected the c.640-801G>A site mutation in the GLA gene. The results were verified using first-generation sequencing technology.
It achieves efficient and accurate detection of GLA gene c.640-801G>A site mutation, reduces detection costs, is suitable for promotion in primary healthcare institutions, and improves detection efficiency and result reliability.
Smart Images
Figure CN121406790A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of gene detection technology, and in particular relates to a primer set, kit and method for detecting genetic susceptibility genes for Fabry cardiomyopathy. Background Technology
[0002] Fabry cardiomyopathy (FC) is a rare X-linked recessive lysosomal storage disorder, one of the main organ involvement manifestations of Fabry disease (α-galactosidase A deficiency). Fabry disease can be divided into classic and late-onset types, and FC can occur in both. Classic type patients usually develop the disease in childhood or adolescence, and in addition to cardiac involvement, they also have multi-system manifestations such as cutaneous angiokeratoma, neurological symptoms (such as acropause and hypohidrosis), and kidney damage. Late-onset patients usually develop the disease in adulthood (30-50 years old), with relatively localized symptoms, and the heart is often the main or even the only affected organ. The clinical manifestations are similar to dilated or hypertrophic cardiomyopathy. The incidence of late-onset FC (especially late-onset FC with predominantly cardiac involvement) is relatively higher than that of Fabry cardiomyopathy. Some studies show that the detection rate of FC in patients with unexplained left ventricular hypertrophy can reach 2%-5%, suggesting that it is not uncommon in certain populations.
[0003] Fabry cardiomyopathy is an X-linked recessive genetic disorder caused by mutations in the GLA gene. The c.640-801G>A mutation is a site-specific mutation in the intron splicing region. Although the mutation occurs in an intron region that does not encode protein, it affects the mRNA splicing process during gene transcription and is considered a pathogenic mutation site in Fabry cardiomyopathy. Detection of this mutation site can assist in carrier screening of family members or in testing suspected Fabry cardiomyopathy patients, further ensuring the accuracy of the test results. Current methods for detecting the GLA gene, the pathogenic gene for Fabry cardiomyopathy, mainly include next-generation sequencing (NGS). However, this technology suffers from complex data interpretation, uneven coverage, and inability to reliably detect certain mutations. It also has limited ability to detect trinucleotide repeats, large deletions, or duplications, and is susceptible to pseudogene interference, potentially leading to false negatives and missed detections of certain types of gene mutations. Therefore, given these shortcomings, developing a gene detection method that accurately identifies mutation types at the c.640-801G>A site in the GLA gene, is simple to operate, low-cost, and suitable for rapid detection is a pressing technical problem that needs to be solved. Summary of the Invention
[0004] (a) Technical problems to be solved In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a primer set, kit and method for detecting genetic susceptibility genes for Fabry cardiomyopathy, which solves the technical problems of complex interpretation of existing Fabry cardiomyopathy gene detection data, uneven coverage, inability to reliably detect certain mutations, limited ability to detect repetitive sequences, and "false negative" results.
[0005] (II) Technical Solution This invention provides a primer set, kit, and method for detecting genetic susceptibility genes for Fabry cardiomyopathy.
[0006] One objective of this invention is to provide a primer set for detecting a genetic susceptibility gene for Fabry cardiomyopathy, wherein the genetic susceptibility gene for Fabry cardiomyopathy is the GLA gene; the mutation site of the GLA gene is c.640-801G>A; the primer set includes a PCR amplification primer set; the PCR amplification primer set includes forward PCR amplification primers and reverse PCR amplification primers; the nucleotide sequence of the forward PCR amplification primers is shown in SEQ ID NO.1; the nucleotide sequence of the reverse PCR amplification primers is shown in SEQ ID NO.2.
[0007] Furthermore, the primer set also includes a Sanger sequencing primer set; the Sanger sequencing primer set includes forward sequencing primers and reverse sequencing primers; the nucleotide sequence of the forward sequencing primer is shown in SEQ ID NO.1; the nucleotide sequence of the reverse sequencing primer is shown in SEQ ID NO.2.
[0008] A second objective of this invention is to provide a detection kit for genetic susceptibility genes of Fabry cardiomyopathy, comprising the detection primer set for genetic susceptibility genes of Fabry cardiomyopathy as described in claim 1 or claim 2.
[0009] Furthermore, the kit includes a PCR amplification reaction system, an enzyme purification reaction system, and a Sanger sequencing reaction system.
[0010] Furthermore, the PCR amplification reaction system includes a PCR amplification primer set, ultrapure water, 2×PCR Buffer, a mixture of dNTPs, and KOD enzyme; the enzyme purification reaction system includes ultrapure water, CIP enzyme, and Exo I enzyme; and the Sanger sequencing reaction system includes a Sanger sequencing primer set, ultrapure water, BigDye Buffer, and BigDye enzyme.
[0011] The third objective of this invention is to provide a method for detecting genetic susceptibility genes for Fabry cardiomyopathy, comprising: S1, extracting template DNA from an in vitro sample, preparing PCR amplification reaction reagents, performing PCR amplification reaction, and obtaining PCR amplification products; separating the PCR amplification products and markers in agarose gel wells by electrophoresis, confirming the band specificity by a gel imaging system, and obtaining verified PCR amplification products; S2, subjecting the verified PCR amplification products to enzyme purification reaction, and obtaining enzyme-purified products; S3, subjecting the enzyme-purified products to Sanger sequencing reaction, and obtaining Sanger sequencing reaction products; S4, separating, purifying, and pre-denaturing the Sanger sequencing reaction products, and obtaining a mixture of denatured single-stranded DNA fragments; S5, separating and detecting the mixture of denatured single-stranded DNA fragments by capillary electrophoresis, and obtaining the genotype of the c.640-801G>A mutation site of the GLA gene by comparative analysis.
[0012] Further, in S3, the purified enzyme product is placed in the Sanger sequencing reaction system; the Sanger sequencing reaction system includes a forward Sanger sequencing reaction system and a reverse Sanger sequencing reaction system; the forward Sanger sequencing reaction system includes forward sequencing primers; the reverse Sanger sequencing reaction system includes reverse sequencing primers.
[0013] Furthermore, the PCR amplification reaction includes PCR amplification primers with a final concentration of 10 μM; the Sanger sequencing reaction includes Sanger sequencing primers with a final concentration of 3.2 μM.
[0014] Furthermore, the Sanger sequencing reaction program includes 95°C for 4 min; 95°C for 15 s; 50°C for 20 s; 60°C for 2 min; 25 cycles; and holding at 4°C.
[0015] Furthermore, the enzyme purification reaction procedure includes 37°C for 50 min; 95°C for 5 min; and holding at 4°C.
[0016] The reference genome version used in this invention is GRCh38 / hg38.
[0017] (III) Beneficial Effects The Fabry cardiomyopathy genetic susceptibility gene detection kit proposed in this invention is complete in components. The primer set and kit involved can accurately detect the c.640-801G>A mutation in the GLA gene. The operation steps are simple, and the requirements for instruments and equipment are low, making it suitable for rapid testing in clinical laboratories, third-party testing institutions, and other scenarios. The kit of this invention also includes PCR amplification reagents for the GLA gene c.640-801G>A and Sanger sequencing reaction reagents. By using a method of amplification reaction, purification, and then sequencing reaction, only a very small amount of in vitro sample is needed to obtain 100% accurate detection results.
[0018] The PCR primer set provided by this invention simultaneously meets the core requirements of PCR amplification and Sanger sequencing, signifying a high degree of uniformity in specificity, efficiency, and experimental compatibility. Through a single design, the dual-fit primer pair must simultaneously meet the stringent requirements of both PCR and Sanger sequencing, ensuring high specificity. It accurately amplifies the fragment containing the target mutation site during PCR, avoiding non-specific amplification, and precisely binds to the template during Sanger sequencing, ensuring clear sequencing signals without interference from stray peaks. Furthermore, the PCR amplification product directly serves as the template for Sanger sequencing. The shared primer pair ensures that the amplified fragment length perfectly matches the sequencing reaction, eliminating the need for additional fragment size adjustments. This reduces primer synthesis costs and validation time, while avoiding experimental errors caused by differences between the two primer sets, reducing experimental complexity and minimizing primer dilution and system preparation steps. Especially in batch detection, it significantly improves experimental efficiency and reduces the probability of operational errors.
[0019] Furthermore, the detection method for Fabry cardiomyopathy genetic susceptibility genes proposed in this invention has a lower detection cost than NGS and is easier to promote in primary healthcare institutions, providing a convenient and reliable technical solution for the early screening of Fabry cardiomyopathy. Attached Figure Description
[0020] Figure 1 This is a reference sequence containing the GLA gene c.640-801G>A mutation site (target base 201bp).
[0021] Figure 2 This is a Sanger sequencing result of the GLA c.640-801G>A site in sample 1 of this embodiment of the invention.
[0022] Figure 3 This is a Sanger sequencing result of the GLA c.640-801G>A site in sample 2 of this embodiment of the invention.
[0023] Figure 4This is a Sanger sequencing result of the GLA c.640-801G>A site in sample 3 of this embodiment of the invention.
[0024] Figure 5 This is a Sanger sequencing result of the GLA c.640-801G>A site in sample 4 of this embodiment of the invention.
[0025] Figure 6 This is a Sanger sequencing result of the GLA c.640-801G>A site in sample 5 of this embodiment of the invention. Detailed Implementation
[0026] To better explain and facilitate understanding of this invention, a detailed description of the invention is provided below with reference to the accompanying drawings and specific embodiments. All raw materials used in this invention are commercially available, and experimental methods not specifically described are based on conventional operating methods known to those skilled in the art.
[0027] This invention proposes a primer set, kit, method, and application for detecting genetic susceptibility genes for Fabry cardiomyopathy. Utilizing a highly targeted "site-specific detection" method, it detects GLA gene c.640-801G>A site mutations known to exist in known families. Furthermore, it provides auxiliary testing for carriers or patients among family members; or verifies c.640-801G>A site mutations in clinically suspected Fabry cardiomyopathy patients, ensuring the accuracy of the test results.
[0028] Example 1
[0029] This embodiment describes the design of a kit containing primer pairs for detecting the GLA gene c.640-801G>A site, which includes: (1) PCR amplification primer set (see Table 1), including the forward primer nucleotide sequence as SEQ ID NO.1 and the reverse primer nucleotide sequence as SEQ ID NO.2;
[0030]
[0031] (2) PCR amplification reagents: 2×PCR Buffer (purchased from TOYOBO Biotechnology Co., Ltd.), dNTP (purchased from TOYOBO Biotechnology Co., Ltd.), PCR amplification primers (final concentration of 10μM) and KOD enzyme (purchased from TOYOBO Biotechnology Co., Ltd.); (3) Reagents for enzyme purification reaction: CIP enzyme (purchased from New England Biolabs), Exo I enzyme (purchased from New England Biolabs); (4) Sanger sequencing reaction reagents: BigDye Buffer (purchased from ABI), BigDye enzyme (purchased from ABI), forward and reverse sequencing primers (final concentration 3.2 μM); (5) Sequencing purification reagents: 125mM EDTA, anhydrous ethanol (purchased from Antere), 75% ethanol (purchased from Hangzhou Optip Biotechnology Co., Ltd.), Hi-Di™ (purchased from ABI).
[0032] Example 2
[0033] This embodiment describes a method for detecting the genotype of the GLA gene c.640-801G>A site using the kit from Example 1, including: (1) PCR amplification reaction and analysis Template DNA was extracted from the in vitro sample, and a PCR premix was prepared (PCR amplification primers were added separately). The PCR amplification reaction was then carried out to obtain the PCR amplification product.
[0034] The proportions of the PCR premix are shown in Table 2.
[0035]
[0036] Dispense 17 μL of the above PCR premix into each well, then add 0.5 μL each of the forward PCR amplification primer and the reverse PCR amplification primer (10 μM), and finally add 2 μL of template DNA. Vortex and briefly centrifuge to mix.
[0037] The PCR amplification reaction was performed using a PCR instrument. The PCR reaction procedure is shown in Table 3, and the PCR amplification products were obtained.
[0038]
[0039] Prepare 2% agarose gels using a DL2000 marker. For each sample, aspirate 2 μL of PCR amplification product and mix with 4 μL of 6×DNA Loading Buffer. Add the marker and sample mixture to the wells of the agarose gel. Then, place the agarose gel into an electrophoresis tank containing 1×TAE. Set the electrophoresis apparatus to 120V and the time to 20-30 minutes.
[0040] Interpreting electrophoresis results in a gel imaging system involves the following steps: First, ensure that the indicator bands in the marker are clearly separated to easily indicate the size of the target band in the sample. If the indicator bands are not completely separated and the size of the target band cannot be determined, extend the electrophoresis time to ensure a clear and interpretable result. Second, confirm that the sample bands are in the correct position and clearly distinguishable. If incorrect positioning, multiple bands, or dragging bands are found, primers must be redesigned before PCR amplification.
[0041] PCR products that pass electrophoresis should be stored at 4°C for later use, and stored at -20°C for long-term storage.
[0042] (2) Enzyme purification reaction Prepare the enzyme purification reaction mixture according to Table 4, and dispense 2 μL into each well; add 9 μL of the corresponding sample's PCR product, so that the total volume of liquid added to each well is 11 μL, vortex and briefly centrifuge to mix.
[0043]
[0044] According to the purification reaction procedure in Table 5, the PCR instrument was used to perform the purification reaction and obtain the purified reaction product.
[0045]
[0046] (3) Sanger sequencing reaction Prepare the Sanger sequencing reaction mixture according to the sequencing reaction system composition shown in Table 6. Dispense the Sanger sequencing reaction mixture into each sample well, with each sample containing both forward and reverse reaction systems and sample wells for corresponding detection. Add 1 μL of forward sequencing primer / 1 μL of reverse sequencing primer (both primer concentrations are 3.2 μM; the nucleotide sequence of the forward sequencing primer is as shown in SEQ ID NO.1, and the nucleotide sequence of the reverse sequencing primer is as shown in SEQ ID NO.2) to each sample well. Then add 2 μL of the purified product obtained from the above enzyme purification reaction to each sample well, vortex, and briefly centrifuge to mix.
[0047]
[0048] Sequencing reactions were performed on a PCR instrument according to the Sanger sequencing reaction procedure (see Table 7) to obtain sequencing reaction products.
[0049]
[0050] (4) Purification of Sanger sequencing reaction products The Sanger sequencing reaction products obtained above were briefly centrifuged. Add 2 μL of 125 mM EDTA to each tube, cap, vortex, and briefly centrifuge. Then add 15 μL of anhydrous ethanol and vortex. Centrifuge at 3700 rpm for 30 min using a plate centrifuge; then briefly centrifuge at 300 rpm inverted position to remove the supernatant. Add the precipitate obtained by centrifugation to 50 μL of pre-chilled 75% ethanol at -20°C, vortex, and centrifuge at 3700 rpm for 15 min. Briefly centrifuge at 300 rpm inverted position to remove the supernatant, and incubate at room temperature for 10 min to allow the ethanol to evaporate completely. Add 10 μL of Hi-Di™ to each tube, vortex thoroughly for 1 min, briefly centrifuge, and incubate for 10 min to allow the sequencing products to completely dissolve. Pre-denature the purified product at 95°C for 5 min, quickly transfer to an ice box, cool at 4°C for 5 min, and then transfer to a 96-well plate for sequencing.
[0051] (5) Sequencing on the machine After preparing the settings and procedures for loading 96-well plates onto the ABI 3500 XL genetic analyzer, the 96-well plates containing the samples were analyzed using the ABI 3500 XL genetic analyzer. After sequencing, the results were converted into a visual spectrum using BioEdit analysis software to obtain the genotype of the GLA c.640-801G>A site in the target region, thereby determining the genetic information of Fabry cardiomyopathy based on this mutation site.
[0052] The reference sequence information of this invention can be found in Figure 1 The target base is 201bp.
[0053] Example 3
[0054] This embodiment describes the detection method of the kit containing PCR amplification primers used in Example 2 to perform genotyping of the GLA c.640-801G>A locus in actual in vitro samples: Genomic DNA was extracted from whole blood from five volunteers, followed by PCR, enzyme purification, and Sanger sequencing. The products were then detected by capillary electrophoresis. Finally, the data were analyzed using relevant software to determine the genotype of the GLA c.640-801G>A locus in each sample. Sequencing results are shown below. Figures 2-6 And Table 8, where the test results of volunteers numbered 1-5 correspond to... Figure 2-6 Meanwhile, the mass spectrometry results were verified by first-generation sequencing. The verification results showed that the results of the method of this invention and the first-generation sequencing method had a 100% agreement rate.
[0055] Based on the above experimental results of detecting the c.640-801G>A site of the GLA gene related to Fabry cardiomyopathy, the kit of this invention, based on PCR amplification reagents and Sanger sequencing reaction reagents, can accurately detect the genotype containing the pathogenic c.640-801G>A variant of the GLA gene. Specifically, male subjects 1 and 4 presented with Fabry cardiomyopathy, and the c.640-801G>A site of the GLA gene was detected as a hemizygous variant; female subjects 2 and 3 presented with asymptomatic and typical Fabry cardiomyopathy symptoms, respectively, and the c.640-801G>A site of the GLA gene was detected as heterozygous; female subject 5 was normal, did not present with Fabry cardiomyopathy symptoms, and was wild-type at the c.640-801G>A site of the GLA gene. The test results are consistent with the X-chromosome recessive inheritance pattern of Fabry cardiomyopathy. The detection method of the kit containing the PCR amplification primer set and Sanger sequencing primer set of the GLA gene c.640-801G>A involved in this invention is accurate and reliable.
[0056]
[0057] In summary, the primer set and kit containing the primer set provided by this invention, based on first-generation sequencing technology, detect the c.640-801G>A site mutation in the GLA gene for Fabry cardiomyopathy. This has significant value for early screening and genetic risk assessment of this disease gene, providing an efficient and reliable gene testing solution. It should be noted that the kit and detection method involved in this invention are not directly used for disease diagnosis, but rather for gene screening or assessment of potential genetic risks based on gene screening.
[0058] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features. These modifications or substitutions, or combinations of technical features in the above embodiments that do not conflict with each other, can be made in accordance with the manner described in the embodiments. These modifications, substitutions or combinations do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.
Claims
1. A primer set for detecting a genetic susceptibility gene for Fabry cardiomyopathy, characterized in that, The genetic susceptibility gene for Fabry cardiomyopathy is the GLA gene. The mutation site of the GLA gene is c.640-801G>A; The primer set includes a PCR amplification primer set; The PCR amplification primer set includes forward PCR amplification primers and reverse PCR amplification primers; The nucleotide sequence of the forward PCR amplification primers is shown in SEQ ID NO.1; The nucleotide sequence of the reverse PCR amplification primers is shown in SEQ ID NO.
2.
2. The primer set for detecting Fabry cardiomyopathy genetic susceptibility genes according to claim 1, characterized in that, The primer set also includes the Sanger sequencing primer set; The Sanger sequencing primer set includes forward sequencing primers and reverse sequencing primers; The nucleotide sequence of the forward sequencing primers is shown in SEQ ID NO.1; The nucleotide sequence of the reverse sequencing primers is shown in SEQ ID NO.
2.
3. A detection kit for genetic susceptibility genes for Fabry cardiomyopathy, characterized in that, Includes the detection primer set for the genetic susceptibility gene of Fabry cardiomyopathy as described in claim 1 or claim 2.
4. The detection kit for Fabry cardiomyopathy genetic susceptibility genes according to claim 3, characterized in that, The kit includes a PCR amplification reaction system, an enzyme purification reaction system, and a Sanger sequencing reaction system.
5. The detection kit for Fabry cardiomyopathy genetic susceptibility genes according to claim 4, characterized in that, The PCR amplification reaction system includes a PCR amplification primer set, ultrapure water, 2×PCR buffer, a mixture of dNTPs, and KOD enzyme; The enzyme purification reaction system comprises ultrapure water, CIP enzyme, and Exo I enzyme; The Sanger sequencing reaction system includes a Sanger sequencing primer set, ultrapure water, BigDye Buffer, and BigDye enzyme.
6. A method for detecting Fabry cardiomyopathy genetic susceptibility genes using the detection primer set for Fabry cardiomyopathy as described in claim 1 or 2, or the detection kit for Fabry cardiomyopathy genetic susceptibility genes as described in any one of claims 3 to 5, characterized in that, include: S1. Extract template DNA from the in vitro sample, prepare PCR amplification reaction reagents, perform PCR amplification reaction, and obtain PCR amplification products; The PCR amplification products were separated by electrophoresis, and the band specificity was confirmed by gel imaging to obtain the validated PCR amplification products. S2. The verified PCR amplification product is subjected to an enzyme purification reaction to obtain the enzyme purified product; S3. Perform Sanger sequencing reaction on the purified enzyme product to obtain the Sanger sequencing reaction product; S4. The Sanger sequencing reaction product is separated, purified, and pre-denatured to obtain a mixture of denatured single-stranded DNA fragments. S5. The denatured single-stranded DNA fragment mixture is separated and detected by capillary electrophoresis, and the genotype of the c.640-801G>A mutation site of the GLA gene is obtained by comparative analysis.
7. The method for detecting Fabry cardiomyopathy genetic susceptibility genes according to claim 6, characterized in that, In S3, the purified enzyme product is placed in the Sanger sequencing reaction system; The Sanger sequencing reaction system includes a forward Sanger sequencing reaction system and a reverse Sanger sequencing reaction system; The forward Sanger sequencing reaction system includes forward sequencing primers; the reverse Sanger sequencing reaction system includes reverse sequencing primers.
8. The method for detecting Fabry cardiomyopathy susceptibility genes according to claim 6, characterized in that, The PCR amplification reaction includes PCR amplification primers with a final concentration of 10 μM; the Sanger sequencing reaction includes Sanger sequencing primers with a final concentration of 3.2 μM.
9. The method for detecting Fabry cardiomyopathy genetic susceptibility genes according to claim 6, characterized in that, The Sanger sequencing reaction program consisted of 95°C for 4 min; 95°C for 15 s; 50°C for 20 s; 60°C for 2 min; 25 cycles; and a 4°C incubation period.
10. The method for detecting Fabry cardiomyopathy susceptibility genes according to claim 6, characterized in that, The enzyme purification reaction procedure includes 37℃ for 50 min; 95℃ for 5 min; and holding at 4℃.