Amplification primer group, kit and method for detecting Alport syndrome pathogenic gene variation

Abstract

The invention provides an amplification primer group, a kit and a method for detecting Alport syndrome virulence gene variation, the amplification primer group amplifies exon coding regions of COL4A3, COL4A4 and COL4A5 genes, and the amplification primer group comprises a plurality of primers with sequences as shown in SEQ ID NO: 1 to SEQ ID NO: 322; the kit comprises the amplification primer group. According to the method, the amplification primer group or the kit is utilized, multiple PCR and high-throughput sequencing are combined, and the variation of the pathogenic gene of the Alport syndrome is detected. The amplification primer group, the kit and the method provided by the invention can be used for detecting exon coding regions of three genes at one time, are high in flux, high in accuracy, economical and efficient, are suitable for various sample types including but not limited to blood samples, saliva samples, throat swabs and the like, are suitable for various high-flux sequencing platforms, and can be used for detecting the exon coding regions of the three genes. The method is beneficial to discussing clinical phenotype characteristics and gene mutation characteristics of the Alport syndrome and discussing whether genotype clinical phenotype association exists or not.

Description

Technical Field

[0001] The present invention relates to the field of gene detection, and in particular to an amplification primer set, a kit and a method for detecting Alport syndrome pathogenic gene mutation. Background Art

[0002] Alport syndrome (AS), also known as eye-ear-kidney syndrome, hereditary nephritis, hereditary progressive nephritis, etc., is characterized by kidney, progressive sensorineural hearing loss and eye involvement. Renal manifestations can progress from microscopic hematuria to proteinuria, progressive renal insufficiency, and eventually end-stage renal disease. Progressive sensorineural hearing loss often occurs in adolescence. Eye changes include anterior lenticule, macular degeneration (white / yellow spots or granulation particles appear in the anterior macular area), corneal endothelial vesicles (polymorphic posterior corneal dystrophy) and recurrent corneal erosions. The main pathogenic genes of this disease are COL4A3, COL4A4 and COL4A5, which encode type IV collagen α3, α4 and α5 chains, respectively. Alport syndrome caused by mutations in the COL4A3 and COL4A4 genes is autosomal dominant or autosomal recessive, accounting for about 15-20%. The main mutation forms are point mutations or small fragment insertion / deletion mutations, and some are caused by gene deletion / duplication. Alport syndrome caused by mutations in the COL4A5 gene is X-linked dominant, accounting for about 80%-85%. Point mutations or small fragment insertion / deletion are the main mutation forms, and some are caused by gene deletion / duplication. Pathogenic gene testing of relevant patients can be carried out to explore the clinical phenotypic characteristics, gene mutation characteristics, and whether there is a genotype-clinical phenotype association of Alport syndrome by analyzing clinical data and genotype characteristics. It can further expand the multi-angle understanding of the clinical manifestations of this type of disease, renal pathological characteristics, and gene mutation types, and can guide clinicians to carry out individualized treatment and accurately judge the prognosis. The existing technology for detecting Alport pathogenic genes has a low sequencing depth. During sequencing, the whole genome or the whole exome is scanned, which is easy to cause problems such as poor sequencing quality and failure to detect specific mutation sites. Summary of the invention

[0003] In view of the above shortcomings of the prior art, the first aspect of the present invention provides an amplification primer set for detecting Alport syndrome pathogenic gene mutations, wherein the amplification primer set comprises multiple primers with sequences as shown in SEQ ID NO: 1 to SEQ ID NO: 322.

[0004] The amplification primer set includes a first primer pool and a second primer pool, the first primer pool includes primers with sequences shown as SEQ ID NO: 1 to SEQ ID NO: 162, and the second primer pool includes primers with sequences shown as SEQ ID NO: 163 to SEQ ID NO: 322.

[0005] The primer pairs each include a forward primer and a reverse primer, the first primer pool includes the 1st primer pair to the 81st primer pair, the second primer pool includes the 82nd primer pair to the 162nd primer pair, the forward primer of the 1st primer pair, the reverse primer of the 1st primer pair to the forward primer of the 81st primer pair, and the reverse primer of the 81st primer pair are respectively shown as SEQ ID NO: 1 to SEQ ID NO: 162 in the sequence listing, the forward primer of the 82nd primer pair, the reverse primer of the 82nd primer pair to the forward primer of the 162nd primer pair, and the reverse primer of the 162nd primer pair are respectively shown as SEQ ID NO: 163 to SEQ ID NO: 322 in the sequence listing, as shown in Table 1.

[0006] A second aspect of the present invention provides a kit for detecting mutations in a pathogenic gene of Alport syndrome, the kit comprising the above-mentioned amplification primer set.

[0007] The present invention uses PCR technology to detect genes COL4A3, COL4A4 and COL4A5. According to the amplification conditions, it can be determined whether COL4A3, COL4A4 and COL4A5 of the sample to be tested have undergone gene mutations. Therefore, the design of primers is the key to the kit of the present invention.

[0008] Since the kit of the present invention adopts PCR technology for detection, the kit may also include other conventional reagents required for PCR, such as DNA polymerase, dNTP mixture, buffer, Index, ddH 2 O. One or more commonly used PCR reaction reagents such as sample genomic DNA extraction reagents. Since such commonly used PCR reagents can be purchased separately from the market or prepared by oneself, the specific reagents that need to be assembled into the kit can be prepared according to the actual needs of the customer, and for convenience, all of them can be assembled into the kit.

[0009] The kit of the present invention may contain independently packaged primer pairs, or may contain a prepared PCR detection solution containing an amplification primer set.

[0010] The PCR detection solution can be prepared by itself, or it can be obtained by directly adding the amplification primer set to a commercially available universal PCR reaction solution without the amplification primer set. For example, the kit can also contain DNA polymerase, dNTP mixture, buffer, index, ddH 2 O. A PCR reaction system can be obtained by adding the amplification primer set of the present invention and the DNA extract of the sample to be tested.

[0011] Preferably, the kit may further contain a positive control, which is a DNA sample containing the expression of genes COL4A3, COL4A4 and COL4A5.

[0012] Preferably, the kit may also contain a negative control, which may be a DNA sample without the expression of the genes COL4A3, COL4A4 and COL4A5.

[0013] The third aspect of the present invention provides the use of the above-mentioned amplification primer set or the above-mentioned kit in preparing a product for detecting the pathogenic gene variation of Alport syndrome, which is used to detect the pathogenic gene variation of Alport syndrome patients.

[0014] A fourth aspect of the present invention provides a method for detecting a genetic variation causing Alport syndrome, wherein the method is a method for non-disease diagnosis purposes and comprises the following steps:

[0015] (1) Obtaining genomic DNA of the sample to be tested;

[0016] (2) performing a first round of multiplex PCR reaction on the genomic DNA using the amplification primer set according to any one of claims 1 to 2 or the kit according to any one of claims 5 to 7;

[0017] (3) performing a second round of linker sequence PCR;

[0018] (4) Sequencing and analyzing the sequencing data.

[0019] The non-disease diagnostic purposes include detecting gene mutations in Alport syndrome patients, detecting the characteristics of gene mutations in Alport syndrome patients, expanding the multi-angle understanding of the types of Alport syndrome gene mutations, and exploring the relationship between the clinical phenotypic characteristics of Alport syndrome patients and the characteristics of gene mutations.

[0020] Furthermore, in step (1), extracting sample genomic DNA is a prior art, and those skilled in the art can prepare it according to existing operating methods, or directly use a ready-made DNA extraction kit and follow the steps of the kit.

[0021] The method of obtaining the genomic DNA of the sample to be tested also includes detecting the concentration and purity of the DNA. The method of detecting the concentration and purity of the DNA is a prior art, and those skilled in the art can select and operate according to the existing operation method. When the purity 260 / 280 ratio is between 1.5 and 2.0 and the concentration is not less than 5ng / μL, it is qualified.

[0022] Furthermore, the step (2) includes the following steps:

[0023] (21) adding the genomic DNA of the sample to be tested, the positive control and / or the negative control to PCR tubes containing the first PCR reaction system, respectively, to obtain corresponding sample reaction tubes, positive reaction tubes and / or negative reaction tubes, and performing a first round of multiplex PCR reaction, wherein the PCR reaction system contains the above-mentioned amplification primer set for detecting the Alport syndrome pathogenic gene variation;

[0024] The first PCR reaction system is shown in Table 1.

[0025] Table 1 The first PCR reaction system

[0026] Pool-1 / Pool-2 reaction system volume IGT-EM808polymerase mixture 10μL Enhancer buffer NB(1N) 3.5μL Enhancer buffer M 2.5μL Primer pool-1 / pool-2 5μL gDNA 40ng <![CDATA[ddH 2 The]]> Make up to 30 μL

[0027] Alternatively, the genomic DNA of the sample to be tested, the positive control and / or the negative control are added to PCR tubes respectively, and the above-mentioned kit for detecting the Alport syndrome pathogenic gene mutation is used;

[0028] (22) The primer pairs in the first primer pool and the primer pairs in the second primer pool respectively perform a first round of multiplex PCR amplification on the genomic DNA of the sample to be tested.

[0029] The conditions of the first round of multiplex PCR reaction were: 95°C for 3 min 30 s, 1 cycle; 98°C for 20 s, 60°C for 4 min, 18 cycles; 72°C for 5 min, 1 cycle.

[0030] Furthermore, the step (3) includes the following steps:

[0031] (31) mixing the product amplified by the first primer pool and the product amplified by the second primer pool in step (2),

[0032] (32) purifying the mixed product,

[0033] (33) preparing a second PCR reaction system;

[0034] (34) Perform a second round of linker sequence PCR.

[0035] The purification in step (32) is a prior art, and those skilled in the art can perform purification according to existing operating methods and adjust the amount of reagents, for example, by using magnetic beads for purification.

[0036] The second PCR reaction system is shown in Table 2.

[0037] Table 2 Second PCR reaction system

[0038] PCR reaction system volume Multiplex PCR products 13.5μL Enhancer buffer M 2.5μL <![CDATA[ddH 2 The]]> 2μL IGT-I5Index(10μM) 1μL IGT-I7Index(10μM) 1μL IGT-EM808polymerase mixture 10μL

[0039] The conditions for the second round of linker sequence PCR reaction were: 95°C for 3 min and 30 s, 1 cycle; 98°C for 20 s, 58°C for 1 min, 72°C for 30 s, 9 cycles; and 72°C for 5 min, 1 cycle.

[0040] Furthermore, the step (4) further comprises purifying and quality checking the amplicon library obtained by the second round of PCR amplification of the adapter sequence before sequencing.

[0041] The purification is a prior art, and those skilled in the art can perform purification according to the existing operation method and adjust the amount of reagents, for example, magnetic beads can be selected for purification.

[0042] The quality inspection is to use Qubit to determine the concentration of the second round of adapter sequence PCR amplicon library, and use Agilent bioanalyzer to measure the library fragment length and purity. If the concentration range of the amplicon library is 5-50ng / μL, the fragment length is distributed between 300-400bp, and there is no impurity peak, the quality inspection is qualified.

[0043] Furthermore, the sequencing in step (4) uses the Illumina NextSeq 550 sequencing platform.

[0044] Furthermore, the result analysis was performed using the bcbio-nextgen process (https: / / github.com / bcbio / bcbio-nextgen) for bioinformatics analysis.

[0045] The beneficial effects of the present invention are:

[0046] (1) High-throughput sequencing technology can sequence hundreds of thousands to millions of DNA molecules at a time, with high throughput and high accuracy. Multiplex PCR technology can add more than two pairs of primers in the same PCR reaction system to amplify multiple nucleic acid fragments at the same time, with strong specificity and short experimental cycle, which can significantly save time and reagent costs. The present invention adopts multiplex PCR technology combined with high-throughput sequencing to design specific primers for Alport syndrome pathogenic genes COL4A3, COL4A4 and COL4A5, which can detect the exon coding regions of the three genes at one time, with high throughput, high accuracy and economic efficiency. This detection method is applicable to a variety of sample types, including but not limited to blood samples, saliva samples, throat swabs, etc., and is applicable to a variety of high-throughput sequencing platforms.

[0047] (2) The present invention is used to detect the mutation of the pathogenic gene of patients with Alport syndrome, which helps to explore whether there is a genotype-clinical-phenotype correlation between the clinical phenotypic characteristics of Alport syndrome and the characteristics of gene mutations, and can further expand the multi-angle understanding of the clinical manifestations of this type of disease, renal pathological characteristics, and gene mutation types. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 is a flow chart of the method of the present invention.

[0049] Figure 2 This is a schematic diagram of the multiplex PCR amplification library construction of the present invention. DETAILED DESCRIPTION

[0050] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0051] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing the specific specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0052] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0053] Example 1 Primer Design of COL4A3, COL4A4 and COL4A5 Pathogenic Genes

[0054] The chromosome location, exon information, sequence information, etc. of COL4A3, COL4A4 and COL4A5 genes were obtained from the NCBI database. A total of 161 pairs of primers were designed for the exon regions of the genes using the primer software. The primer length was between 20-30 nt and the amplified fragment length was between 150-300 bp. After verification, specific primers with good amplification characteristics were obtained. Since the distance between the primer amplification regions was too close, the primer amplification was performed in two tubes, namely the first primer pool and the second primer pool. The first primer pool contained the first primer pair to the 81st primer pair, a total of 81 pairs of specific primers, and the second primer pool contained the 82nd primer pair to the 161st primer pair, a total of 80 pairs of specific primers. The above-mentioned specific primers, primer sequence numbers, primer sequences and amplified fragment lengths are listed in Table 1.

[0055] Table 3 Primer information

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063] Example 2 Clinical sample detection

[0064] This example uses 13 clinical blood samples to illustrate a method for detecting COL4A3, COL4A4 and COL4A5 gene mutations based on multiplex PCR and high-throughput sequencing technology, including the following steps:

[0065] (1) The genomic DNA of 12 samples was extracted using a finished kit according to the operating procedures of the finished kit, and the concentration and purity were tested. The purity 260 / 280 ratio was between 1.5 and 2.0, and the concentration was not less than 5 ng / μL.

[0066] (2) First round of multiplex PCR reaction:

[0067] The specific amplification primer set of the present invention and other related reagents are used to prepare a first PCR reaction system. The first primer pool and the second primer pool are used to perform PCR amplification on the genomic DNA to be tested. The first PCR reaction system is shown in Table 1.

[0068] Table 2 The first PCR reaction system

[0069] Pool-1 / Pool-2 reaction system volume IGT-EM808polymerase mixture 10μL Enhancer buffer NB(1N) 3.5μL Enhancer buffer M 2.5μL Primer pool-1 / pool-2 5μL gDNA 40ng <![CDATA[ddH 2 The]]> Make up to 30 μL

[0070] The first round of multiplex PCR reaction program was: 95°C for 3 min 30 s, 1 cycle; 98°C for 20 s, 60°C for 4 min, 18 cycles; 72°C for 5 min, 1 cycle.

[0071] (3) Second round of linker sequence PCR:

[0072] The multiplex PCR products of the first round of the first primer pool and the second primer pool were mixed and purified by magnetic beads, and the second PCR reaction system shown in Table 2 was prepared for terminal modification and index connection.

[0073] Table 3 Second PCR reaction system

[0074] PCR reaction system volume Multiplex PCR products 13.5μL Enhancer buffer M 2.5μL <![CDATA[ddH 2 The]]> 2μL IGT-I5Index(10μM) 1μL IGT-I7Index(10μM) 1μL IGT-EM808polymerase mixture 10μL

[0075] The reaction program of the second round of linker sequence PCR was as follows: 95°C for 3 min and 30 s, 1 cycle; 98°C for 20 s, 58°C for 1 min, 72°C for 30 s, 9 cycles; and 72°C for 5 min, 1 cycle.

[0076] (4) Purification of amplified library

[0077] ① Add 27 μL of AMPure XP magnetic beads equilibrated at room temperature to 30 μL of PCR reaction system and gently pipette

[0078] Mix 20 times;

[0079] ②After incubation at room temperature for 5 minutes, place the PCR tube on a magnetic stand for 3 minutes;

[0080] ③Completely remove the supernatant, remove the PCR tube from the magnetic rack, add 50 μL YF buffer B to the tube, and gently pipette

[0081] Mix by slowly pumping 20 times;

[0082] ④After incubation at room temperature for 5 minutes, place the PCR tube on the magnetic rack for 3 minutes;

[0083] ⑤ Remove the supernatant, place the PCR tube on the magnetic rack, add 180 μL of 80% ethanol solution into the tube, and let it stand for 30 seconds;

[0084] Let stand at room temperature for 3 min to allow the residual ethanol to evaporate completely;

[0085] ⑥ Remove the PCR tube from the magnetic rack, add 24 μL Nuclease-free water or 1×TE buffer (pH 8.0), and transfer

[0086] ⑦ Gently pipette to resuspend the magnetic beads to avoid bubbles and let stand at room temperature for 2 minutes;

[0087] ⑧Put the PCR tube back on the magnetic rack and let it stand for 3 minutes;

[0088] Use a pipette to take out 20 μL of supernatant and transfer it to a new PCR tube, which is the purified multiplex PCR library.

[0089] (5) Library quality inspection and sequencing

[0090] The concentration of the library after magnetic bead purification was determined using Qubit, and the normal library concentration range was 5-50 ng / μL. The length and purity of the library fragments were measured using an Agilent bioanalyzer. If the fragment length was between 300-400 bp and there were no impurity peaks, the quality inspection was qualified and the Illumina NextSeq 550 sequencing platform was used for sequencing.

[0091] (6) Data analysis and test results

[0092] After the sequencing data was downloaded, the bcbio-nextgen process (https: / / github.com / bcbio / bcbio-nextgen) was used for bioinformatics analysis to obtain the variation information of COL4A3, COL4A4, and COL4A5 genes of 13 clinical samples. The specific results are shown in Table 4. Pathogenic variants were analyzed in all 13 cases, which was consistent with the results obtained based on whole exome sequencing. Clinicians can analyze clinical data and genotype characteristics to explore the clinical phenotype characteristics of Alport syndrome, gene mutation characteristics, and whether there is a genotype-clinical phenotype association, which can guide patients to carry out individualized treatment and accurately judge prognosis.

[0093] Table 4 Test results

[0094] sample Gene Transcript number Mutations type 100004-01 COL4A5 NM_033380.3 c.4962G>A Hemizygous 100098-01 COL4A3 NM_000091.5 c.600_603dup Heterozygous 100104-01 COL4A5 NM_033380.3 c.1117C>T Hemizygous 100112-01 COL4A5 NM_033380.3 c.584G>A Hemizygous 100235-01 COL4A3 NM_000091.5 c.2990G>A Homozygous 210927-01 COL4A5 NM_033380.3 c.1303G>C Hemizygous 207700-01 COL4A5 NM_033380.3 c.4528+1G>C Hemizygous 210901-01 COL4A5 NM_033380.3 c.4706G>A Hemizygous 210902-01 COL4A5 NM_033380.3 COL4A5 Hemizygous 210906-01 COL4A5 NM_033380.3 c.81+2dup Hemizygous 210925-01 COL4A5 NM_033380.3 c.4822-1G>T Hemizygous 210921-01 COL4A5 NM_033380.3 c.703C>T Hemizygous 3010162 COL4A4 NM_000092.5 c.1724G>A Heterozygous 3010162 COL4A4 NM_000092.5 c.3636_3637del Heterozygous

[0095] Example 3 Specificity experiment of amplification primer set

[0096] Experimental methods:

[0097] This example uses samples from 4 patients with chronic kidney disease caused by other genes that were clinically diagnosed and confirmed by whole exome sequencing (Table 5), and adopts the experimental process in Example 2 to detect COL4A3, COL4A4 and COL4A5 gene mutations to confirm the specificity of the amplification primer set.

[0098] Table 5

[0099]

[0100] Experimental results: The sequencing and analysis results showed that the four clinical samples failed to detect the known pathogenic variants listed in Table 5. Therefore, this amplification primer set can only detect pathogenic variants of COL4A3, COL4A4, and COL4A5 genes but cannot detect pathogenic variants of other genes. Therefore, the amplification primer set and method of the present invention have good specificity.

[0101] Example 4 Sensitivity test of amplification primer set

[0102] Experimental methods:

[0103] Experiment 4-1 used a patient with Alport syndrome (100235-01) diagnosed by clinical examination and genetic testing, and diluted the DNA concentration to 0.25, 0.5, 1.0, 2 and 4 ng / μL, and used the experimental process in Example 2. The obtained sequencing data was quality controlled to explore the effects of different DNA concentrations on indicators such as average sequencing and uniformity of sequencing data.

[0104] Experiment 4-2 Based on the results of 4-1, a patient with Alport syndrome (100235-01) diagnosed by clinical examination and genetic testing was used, and the DNA concentration was diluted to 0.5, 1.0 and 2 ng / μL. The experimental process in Example 2 was repeated 20 times for each concentration to detect COL4A3, COL4A4 and COL4A5 gene mutations. The sensitivity of the amplification primer set was confirmed by whether the COL4A3c.2990G>A mutation and the zygotic type could be detected.

[0105] Experimental results:

[0106] The results of Experiment 4-1 showed that at concentrations of 0.5 ng / μL and above, the sequencing data had good uniformity and on-target rate, so the test concentrations of Experiment 3-2 were determined to be 0.5, 1.0, and 2 ng / μL.

[0107] The results of Experiment 4-2 showed that at a concentration of 1.0 ng / μL and above, the COL4A3c.2990G>A mutation was correctly detected in each of the 20 experiments, and the zygotic type was correctly determined, and 100% of the targeted intervals in 19 or 20 experimental results could reach a 20X sequencing depth. The results are shown in Table 6. Only a concentration of 1.0 ng / μL is required to detect the Alport syndrome pathogenic gene mutation, so the amplification primer set and method of the present invention have high sensitivity.

[0108] Table 6

[0109]

[0110]

[0111] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

Claims

1. An amplification primer set for detecting mutations in Alport syndrome pathogenic genes, wherein the amplification primer set amplifies exon coding regions of COL4A3, COL4A4 and COL4A5 genes, and the amplification primer set comprises multiple sequences of primers shown in SEQ ID NO: 1 to SEQ ID NO:

322.

2. The amplification primer set according to claim 1, It is characterized in that The amplification primer set includes a first primer pool and a second primer pool, the first primer pool includes primers with sequences shown as SEQ ID NO: 1 to SEQ ID NO: 162, and the second primer pool includes primers with sequences shown as SEQ ID NO: 163 to SEQ ID NO:

322.

3. A kit for detecting mutations in the Alport syndrome gene. It is characterized in that Comprising the amplification primer set according to claim 1.

4. The kit according to claim 3, It is characterized in that The kit also includes DNA polymerase, dNTP mixture, buffer, Index, ddH 2 O.

5. The kit according to claim 3, It is characterized in that The kit also contains a positive control and / or a negative control.

6. Use of the amplification primer set according to any one of claims 1-2 and the kit according to any one of claims 3-5 in the preparation of a product for detecting Alport syndrome pathogenic gene mutations.

7. A method for detecting a genetic variation causing Alport syndrome, wherein the method is not intended for disease diagnosis. It is characterized in that The steps include: (1) Obtaining genomic DNA of the sample to be tested; (2) performing a first round of multiplex PCR reaction on the genomic DNA using the amplification primer set according to any one of claims 1 to 2 or the kit according to any one of claims 3 to 5; (3) performing a second round of linker sequence PCR; (4) Sequencing and analyzing the sequencing data.

8. The method for detecting Alport syndrome pathogenic gene variation according to claim 7, Features: The step (2) comprises: The primer pairs in the first primer pool and the primer pairs in the second primer pool respectively perform a first round of multiplex PCR amplification on the genomic DNA of the sample to be tested; And / or; The conditions of the first round of multiplex PCR reaction are: 95°C 3min 30s, 1 cycle; 98°C 20s, 60°C 4min, 18 cycles; 72°C 5min, 1 cycle.

9. The method for detecting Alport syndrome pathogenic gene variation according to claim 7, It is characterized in that The step (3) comprises: mixing the amplification products in the first primer pool and the amplification products in the second primer pool; And / or, the conditions for the second round of linker sequence PCR reaction are: 95°C for 3 min and 30 s, 1 cycle; 98°C for 20 s, 58°C for 1 min, 72°C for 30 s, 9 cycles; 72°C for 5 min, 1 cycle.

10. The method for detecting Alport syndrome pathogenic gene variation according to claim 7, It is characterized in that Step (4) further comprises purifying and quality checking the amplicon library obtained by the second round of PCR amplification of the adapter sequence before sequencing. Preferably, the quality check is to use Qubit to determine the concentration of the amplicon library of the second round of PCR amplicon of the adapter sequence, and to use Agilent bioanalyzer to measure the fragment length and purity of the library. If the concentration of the amplicon library is in the range of 5-50 ng / μL, the fragment length is distributed between 300-400 bp, and there is no impurity peak, the quality check is qualified.

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