Amplification primer group of renal cyst disease gene PKD1, mutation detection method and application

Abstract

The invention belongs to the technical field of genes, and particularly relates to an amplification primer group of a renal cyst disease gene PKD1, a mutation detection method and application, the amplification primer group comprises four pairs of primers for specifically amplifying first to 34 exons of the PKD1 gene; the mutation detection method comprises the following steps: acquiring genome DNA (Deoxyribose Nucleic Acid); carrying out ultra-long fragment PCR amplification on the genome DNA through the primers, carrying out electrophoresis detection, and purifying a target fragment; mixing the purified amplification product with genome DNA in proportion, constructing a sequencing library, and performing high-throughput sequencing through target area capture; and analyzing sequencing data, and screening pathogenic PKD1 gene mutation. Compared with the prior art, the PKD1 ultra-long fragment PCR amplification primer and the renal cyst disease gene PKD1 mutation detection method have the advantages that true and false genes of PKD1 can be distinguished, the diagnosis accuracy is greatly improved, the operation process is simplified, the timeliness is improved, and the detection flux is improved.

Description

Technical Field

[0001] The present invention belongs to the field of gene technology, and particularly relates to an amplification primer set for the gene PKD1 of polycystic kidney disease, a mutation detection method and applications thereof. Background Art

[0002] Polycystic kidney disease (PKD) is a common hereditary chronic kidney disease, mainly manifested as cysts of different sizes in both kidneys (occasionally unilateral), with the cysts filled with fluid, eventually leading to kidney enlargement and deformation. Polycystic kidney disease can be divided into autosomal dominant polycystic kidney disease (ADPKD) and autosomal recessive polycystic kidney disease (ARPKD). The incidence of autosomal dominant polycystic kidney disease is about 1 / 1000 - 1 / 500; the incidence of autosomal recessive polycystic kidney disease is about 1 / 40000 - 1 / 20000. Polycystic kidney disease can occur at any age and in any race, and is one of the main causes of renal failure. Autosomal dominant polycystic kidney disease is the most common. Currently, more than 40 genes have been reported to be related to ADPKD, including PKD1, PKD1, ALG5, ALG9, DNAJB11, GANAB, IFT140, etc. Among them, mutations in the PKD1 gene and PKD2 account for more than 85% of polycystic kidney disease. Among them, the abnormal genes of 90% of ADPKD patients are located on the short arm of chromosome 16, called the ADPKD1 gene, which encodes polycystin-1 (PC1). PC1 is a transmembrane protein widely distributed on the plasma membrane and cilia of renal tubular epithelial cells. It is one of the important proteins for maintaining the normal structure and function of renal tubules, and its mutation will lead to the occurrence of polycystic kidney. The base sequence of the PKD2 gene is relatively simple. In contrast, the PKD1 gene sequence is relatively complex, with six pseudogenes PKD1P1 - PKD1P6, and the first 33 exons have 97.7% DNA sequence homology with the pseudogenes, and the GC content in some regions exceeds 70%, making comprehensive PKD1 mutation screening challenging.

[0003] There are many methods for PKD1 gene detection. Currently, the commonly used methods are: PCR+Sanger sequencing, exon capture sequencing (NGS), or third-generation sequencing. For PCR-Sanger sequencing, several long-fragment PCRs are first used to amplify each region of the PKD1 gene, and then each exon is individually PCR-amplified and sequenced. On the one hand, most of these methods do not include exon 1. On the other hand, the operation is relatively complex, requiring a high workload and cost, and it is difficult to specifically amplify the PKD1 true gene. The accuracy of the results will be interfered by the pseudogene sequence; due to the existence of highly homologous pseudogenes and regions rich in GC content, the primer Panel designed by the conventional exon capture high-throughput sequencing (NGS) method has a very low coverage rate (<70%) on the PKD1 gene exons, failing to achieve the expected purpose; the cost of detecting PKD1 by the third-generation sequencing method is relatively high.

[0004] Therefore, there is still a need in the art to seek a new method for detecting gene mutations related to polycystic kidney disease, improving the diagnostic accuracy rate, simplifying the operation process, enhancing the timeliness, and increasing the detection throughput. Summary of the Invention

[0005] (I) Technical Problems to be Solved

[0006] The object of the present invention is to provide an amplification primer set for the kidney cyst disease gene PKD1, a mutation detection method and application thereof to solve at least one of the above problems, so as to solve the problems in the prior art that the PKD1 gene sequencing operation is complex and costly, and it is difficult to specifically amplify the PKD1 true gene, and the accuracy of the results will be interfered by the pseudogene sequence, and achieve the effects of improving the diagnostic accuracy rate, simplifying the operation process, enhancing the timeliness, and increasing the detection throughput.

[0007] (II) Technical Solutions

[0008] The object of the present invention is achieved through the following technical solutions:

[0009] The present invention relates to specific primers for ultra-long fragment PCR amplification of the kidney cyst disease gene PKD1 and a method for detecting mutations in the kidney cyst disease gene PKD1. The method uses ultra-long chain PCR technology to amplify the PKD1 gene. After purification and mixing of the PCR products, a second-generation sequencing library is constructed. The library is subjected to liquid hybridization capture using a Panel related to kidney cyst disease, and sequencing is performed using a DNBSEQ-T7 sequencer. Kidney cyst disease mutation information, including PKD1 gene mutation information, is obtained through bioinformatics analysis.

[0010] The first aspect of the present invention discloses an amplification primer set for the kidney cyst disease gene PKD1, and the amplification primer set includes 4 pairs of primers for specifically amplifying exons 1 to 34 of the PKD1 gene:

[0011] Primers for amplifying exon 1 of the PKD1 gene, with the forward primer as shown in SEQ ID NO:1 and the reverse primer as shown in SEQ ID NO:2;

[0012] Primers for amplifying exons 2 - 14 of the PKD1 gene, with the forward primer as shown in SEQ ID NO:3 and the reverse primer as shown in SEQ ID NO:4;

[0013] Primers for amplifying exons 15 - 21 of the PKD1 gene, with the forward primer as shown in SEQ ID NO:5 and the reverse primer as shown in SEQ ID NO:6;

[0014] Primers for amplifying exons 22 - 34 of the PKD1 gene, with the forward primer as shown in SEQ ID NO:7 and the reverse primer as shown in SEQ ID NO:8.

[0015] Furthermore, the size of the PDK1 gene amplicon 1 fragment amplified by the primers for amplifying exon 1 of the PKD1 gene is 2813bp;

[0016] The size of the PDK1 gene amplicon 2 fragment amplified by the primers for amplifying exons 2 - 14 of the PKD1 gene is 8573bp;

[0017] The size of the PDK1 gene amplicon 3 fragment amplified by the primers for amplifying exons 15 - 21 of the PKD1 gene is 7092bp;

[0018] The size of the PDK1 gene amplicon 4 fragment amplified by the primers for amplifying exons 22 - 34 of the PKD1 gene is 8460bp.

[0019] Furthermore, the GC content of the primers of SEQ ID NO:1 - 8 is 40% - 60%, and there is no repeated sequence of more than 4 consecutive identical bases. Primer design needs to meet GC balance and avoid repeated sequences to ensure annealing specificity.

[0020] Furthermore, by comparing the base sequences of the PKD1 true gene and 6 pseudogenes through BLAST, the homologous sequence fragments of the 6 pseudogene sequences relative to the PKD1 true gene sequence and the different sites between the pseudogene sequences and the PKD1 true gene sequence on these homologous sequence fragments are obtained, and then 4 pairs of PKD1 true gene - specific PCR amplification primers are designed.

[0021] Furthermore, the average coverage depth of the amplified exons 1 - 34 reaches 450×, and the coverage rate of all exons is 100%.

[0022] The second aspect of the present invention discloses a method for detecting mutations in the gene PKD1 for renal cyst disease, comprising the following steps:

[0023] (1) Obtain genomic DNA;

[0024] (2) Perform ultra-long fragment PCR amplification on the genomic DNA in step (1) using the amplification primer set, detect by electrophoresis and purify the target fragment;

[0025] (3) Mix the purified amplification product obtained in step (2) with genomic DNA in proportion, construct a sequencing library and perform high-throughput sequencing through target region capture;

[0026] (4) Analyze the sequencing data and screen for pathogenic PKD1 gene mutations.

[0027] Further, in step (2), the ultra-long fragment amplification reaction conditions are as follows: pre-denaturation at 98°C for 3 min, followed by 35 cycles, each cycle consisting of denaturation at 98°C for 15 s, annealing at 68°C for 20 s, extension at 72°C for 9 min, and finally thorough extension at 72°C for 9 min, and store at 4°C.

[0028] Further, in step (2), the electrophoresis is agarose gel electrophoresis. After electrophoresis, detect the PCR product and recover the PCR band of the target size to purify the target fragment.

[0029] Further, in step (3), the addition amount of the purified amplification product is calculated according to the following formula:

[0030] Purified amplification product genomic quality (ng) = (X * N * 660 * 10 -6 ) * 10, where X is the femtomole (fmol) number of the recovered fragment; N is the length of the recovered fragment, unit: bp; 660 * 10 -6 is the average molecular weight (MW) of each pair of bases, unit: ng / fmol;

[0031] In the above formula, the purified amplification product genomic is mixed into the genome in a 10-fold amount to reduce the interference of pseudogenes in the genome on the true gene.

[0032] Further, in step (3), perform quality inspection after constructing the second-generation sequencing library.

[0033] Further, in step (3), use a Panel related to renal cyst disease to perform liquid hybridization capture of target region capture on the library.

[0034] Further, in step (4), identify pathogenic PKD1 gene mutations through sequence alignment, variant detection and functional annotation.

[0035] Further, the method for detecting mutations in the polycystic kidney disease gene PKD1 includes the following steps:

[0036] (1) Obtain genomic DNA;

[0037] (2) Use the amplification primer set of the polycystic kidney disease gene PKD1 of the present invention to perform a PCR amplification reaction on the obtained genomic DNA, and use agarose gel electrophoresis to detect the PCR products and recover the PCR bands of the target size;

[0038] (3) After quantifying the recovered products of the PCR bands of the target size, mix them into a fixed amount of genomic DNA according to a certain ratio, then construct a next-generation sequencing library, use a Panel related to polycystic kidney disease to perform liquid hybridization capture on the library, and perform high-throughput sequencing after enriching the captured products;

[0039] (4) Align the DNA sequence fragments obtained by single-sample sequencing to the reference target gene through bioinformatics analysis, extract SNVs (single nucleotide variations) and Indels (insertions and deletions), perform mutation function annotation, and exclude polymorphic variations to obtain the polycystic kidney disease mutation information of the specimen.

[0040] Further, the library can be quantitatively quality-controlled by using a fluorescence quantifier such as Qubit TM 4.0 Fluorometer or quantitative PCR and other methods.

[0041] Further, the library can use (Agilent), Qsep100 (Bioptic) and other fragment analysis instruments to perform quality inspection on the library fragment distribution.

[0042] The third aspect of the present invention discloses the application of the amplification primer set of the polycystic kidney disease gene PKD1 as described above in the detection of mutations in the polycystic kidney disease gene PKD1.

[0043] Further, the amplification primer set of the polycystic kidney disease gene PKD1 can be used to prepare a diagnostic kit for autosomal dominant polycystic kidney disease.

[0044] (III) Beneficial effects

[0045] Compared with the prior art, the present invention has the following advantages:

[0046] (1) When constructing the amplification primer set for the gene PKD1 of renal cyst disease in the present invention, the differential sites in the homologous regions between PKD1 and 6 pseudogenes were accurately located through BLAST alignment. When designing the 3'-end of the primers, the homologous regions were avoided and mismatched bases of the pseudogenes were introduced. Moreover, the GC content in the amplification primer set was 40%-60%, and there was no repetitive sequence with more than 4 consecutive identical bases. Using the amplification primer set constructed in the present invention, the influence of 6 pseudogenes of PKD1 on the binding of exon capture sequencing (NGS) Panel and the design of Sanger sequencing primers was solved, and the sensitivity of PKD1 gene detection was improved.

[0047] (2) In the method for detecting mutations in the gene PKD1 of renal cyst disease in the present invention, by specifically amplifying the true gene fragment of PKD1, the proportion of the target sequence was increased from 0.001% to 5%, and the library was subjected to liquid hybridization capture using the Panel of renal cyst disease to achieve the effect of double enrichment. After the capture products were enriched, high-throughput sequencing was performed, and mutations of renal cyst disease were extracted through bioinformatics, greatly improving the diagnostic accuracy, simplifying the operation process, improving the timeliness, and increasing the detection throughput.

[0048] (3) In the method for detecting mutations in the gene PKD1 of renal cyst disease in the present invention, the PCR amplification products were added according to the formula calculation. Utilizing the complementarity of two DNA sources, the high GC / complex regions that were easily lost during the construction of the sequencing library were forcibly covered by the amplification products, and the natural fragmentation characteristics were retained by retaining the original DNA, preventing uneven coverage caused by amplification bias.

[0049] (4) The ultra-long fragment PCR amplification primer set for the gene PKD1 of renal cyst disease constructed in the present invention can be used to detect mutations in the gene PKD1 of renal cyst disease, and can also be used to prepare a diagnostic kit for autosomal dominant polycystic kidney disease. It has high detection sensitivity, simple operation, low cost, and is easy to implement. Brief Description of the Drawings

[0050] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0051] Figure 1 It is a base sequence comparison diagram of the PKD1 exon and pseudogene in the region of 1-34. The positions marked by the boxes are the specific sites of the true gene; among them Figure 1 (a) shows the sequence alignment of exon 1 of the PKD1 gene between the true gene and the pseudogene; Figure 1(b) shows the sequence alignment of the true and false genes of exons 2-14 in the PKD1 gene; Figure 1 (c) shows the sequence alignment of the true and false genes of exons 15-21 in the PKD1 gene; Figure 1 (d) shows the sequence alignment of the true and false genes of exons 22-34 in the PKD1 gene.

[0052] Figure 2 This is the electrophoresis quality inspection map of the long fragment PCR amplification products of four amplicons of PKD1 in the examples of the present invention, where Figure 2 (a) is the electrophoresis map of the long fragment PCR amplification products of four amplicons of PKD1. From left to right, they are Marker (Takara, 3426A), exon 1 amplification product (amplicon 1), exon 2-14 amplification product (amplicon 2), exon 15-21 amplification product (amplicon 3), exon 22-34 amplification product (amplicon 4), Marker (Takara, 3426A); Figure 2 (b) shows the bp length corresponding to each band of Marker (Takara, 3426A) in the electrophoresis map.

[0053] Figure 3 This is the library quality inspection map constructed using the panel related to renal cyst disease in the examples of the present invention.

[0054] Figure 4 This is the coverage depth and coverage rate map obtained by sequencing the library constructed using the panel related to renal cyst disease in the examples of the present invention through MGIT7 and bioinformatics analysis and statistics.

[0055] Figure 5 This is the IGV view of the gene detection results after amplifying the PKD1 gene using the amplification primer set of the present invention in the examples. The positions marked by the boxes are the specific sites of the true gene; where Figure 5 (a) shows the gene detection situation in the exon 1 region of the PKD1 gene; Figure 5 (b) shows the gene detection situation in the exon 2-14 region of the PKD1 gene; Figure 5 (c) shows the gene detection situation in the exon 15-21 region of the PKD1 gene; Figure 5 (d) shows the gene detection situation in the exon 22-34 region of the PKD1 gene.

[0056] Figure 6 This is the Sanger sequencing verification result of the specific sites of the true gene in the gene obtained after amplifying the PKD1 gene using the amplification primer set of the present invention in the examples. The positions marked by the boxes are the specific sites of the true gene; where Figure 6 (a) shows the sequencing verification result in the exon 1 region of the PKD1 gene;Figure 6 (b) is the sequencing verification result of the region of exons 2-14 in the PKD1 gene; Figure 6 (c) is the sequencing verification result of the region of exons 15-21 in the PKD1 gene; Figure 6 (d) is the sequencing verification result of the region of exons 22-34 in the PKD1 gene. Detailed implementation manners

[0057] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0058] The following specific examples illustrate the implementation manners of the present application. Those skilled in the art can easily understand other advantages and effects of the present application from the content disclosed in this specification. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. The present application can also be implemented or applied through other different specific implementation manners. Various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present application. It should be noted that, without conflict, the following embodiments and the features in the embodiments can be combined with each other. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present application without creative efforts belong to the scope of protection of the present application.

[0059] It should be noted that the following describes various aspects of the embodiments within the scope of the appended claims. It should be obvious that the aspects described herein can be embodied in a wide variety of forms, and any specific structure and / or function described herein is illustrative only. Based on the present application, those skilled in the art should understand that one aspect described herein can be implemented independently of any other aspect, and two or more of these aspects can be combined in various ways. For example, any number and aspects described herein can be used to implement the device and / or practice the method. In addition, this device and / or this method can be implemented using other structures and / or functions in addition to one or more of the aspects described herein.

[0060] In addition, in the following description, specific details are provided for the purpose of thorough understanding of the examples. However, those skilled in the art will understand that the examples can be practiced without these specific details.

[0061] Unless otherwise specified, the raw materials or devices used in the following embodiments are all commercially available raw materials or conventional experimental devices.

[0062] The following describes the technical solutions provided by the embodiments of the present application with reference to the accompanying drawings.

[0063] The present invention provides a set of amplification primers for the gene PKD1 related to renal cyst disease, which includes 4 pairs of primers. Specifically, by comparing the base sequences of the true gene of PKD1 and 6 pseudogenes through BLAST, the homologous sequence fragments of the 6 pseudogene sequences relative to the true gene sequence of PKD1 and the different sites between the pseudogene sequences and the true gene sequence on these homologous sequence fragments are obtained, and then 4 pairs of specific PCR amplification primers for the true gene of PKD1 are designed.

[0064] The comparison of the base sequences of the exons of PKD1 and the pseudogenes within the region of 1 - 34 is as Figure 1 shown, and the positions marked by the boxes are the specific sites of the true gene; Figure 1 (a) shows the sequence alignment of exon 1 of the PKD1 gene between the true gene and the pseudogene; Figure 1 (b) shows the sequence alignment of exons 2 - 14 of the PKD1 gene between the true gene and the pseudogene; Figure 1 (c) shows the sequence alignment of exons 15 - 21 of the PKD1 gene between the true gene and the pseudogene; Figure 1 (d) shows the sequence alignment of exons 22 - 34 of the PKD1 gene between the true gene and the pseudogene.

[0065] The amplification primer set includes 4 pairs of primers for specifically amplifying exons 1 to 34 of the PKD1 gene:

[0066] The primer for amplifying exon 1 of the PKD1 gene, its forward primer is as shown in SEQ ID NO:1, and its reverse primer is as shown in SEQ ID NO:2. The size of the amplified fragment of the PDK1 gene amplicon 1 is 2813bp;

[0067] The primer for amplifying exons 2 - 14 of the PKD1 gene, its forward primer is as shown in SEQ ID NO:3, and its reverse primer is as shown in SEQ ID NO:4. The size of the amplified fragment of the PDK1 gene amplicon 2 is 8573bp;

[0068] The primer for amplifying exons 15 - 21 of the PKD1 gene, its forward primer is as shown in SEQ ID NO:5, and its reverse primer is as shown in SEQ ID NO:6. The size of the amplified fragment of the PDK1 gene amplicon 3 is 7092bp;

[0069] The primer for amplifying exons 22 - 34 of the PKD1 gene, its forward primer is as shown in SEQ ID NO:7, and its reverse primer is as shown in SEQ ID NO:8. The size of the amplified fragment of the PDK1 gene amplicon 4 is 8460bp.

[0070] The primers of SEQ ID NO: 1-8 have a GC content of 40%-60% and do not have a repetitive sequence of more than 4 consecutive identical bases.

[0071] The amplification primer set for the polycystic kidney disease gene PKD1 can be used for the mutation detection of the polycystic kidney disease gene PKD1. The specific method includes:

[0072] (1) Obtain genomic DNA;

[0073] (2) Use the amplification primer set of the polycystic kidney disease gene PKD1 of the present invention to perform a PCR amplification reaction on the obtained genomic DNA, use agarose gel electrophoresis to detect the PCR product and recover the PCR band of the target size;

[0074] (3) After quantifying the recovered product of the PCR band of the target size, mix it into a fixed amount of genomic DNA in a certain amount. The calculation formula for the added amount of the recovered product is: Purified amplified product genomic mass (ng) = (X * N * 660 * 10 -6 ) * 10, where X is the femtomole (fmol) number of the recovered fragment; N is the length of the recovered fragment, unit: bp; 660 * 10 -6 is the average molecular weight (MW) of each pair of bases, unit: ng / fmol; then construct a next-generation sequencing library, use a Panel related to polycystic kidney disease to perform liquid hybridization capture on the library, and perform high-throughput sequencing after enriching the captured product;

[0075] (4) Align the DNA sequence fragments obtained by single-sample sequencing to the reference target gene through bioinformatics analysis, extract SNVs (single nucleotide variations) and Indels (insertions and deletions), perform mutation function annotation, and exclude polymorphic variations to obtain the polycystic kidney disease mutation information of this specimen.

[0076] Example 1

[0077] Refer to Figures 1 to 6 , this example conducts mutation detection of the polycystic kidney disease gene PKD1 on the samples of the subjects. The specific process is as follows:

[0078] 1. Genomic DNA extraction

[0079] Collect 2 ml of peripheral blood from the subject, and use the Meiji Blood DNA Extraction Kit (column method) to extract genomic gDNA. Use a Nanodrop and Qubit 4.0 instrument to measure the DNA purity and concentration.

[0080] 2. PKD1 ultra-long fragment PCR amplification

[0081] (1) Use 2X Phanta Max Master Mix (Vazyme P515) for PKD1 ultra-long fragment PCR amplification. The primer sequences are shown in Table 1, the reaction system is shown in Table 2, and the reaction program is shown in Table 3.

[0082] Table 1: Primers for PKD1 ultra-long fragment PCR amplification

[0083]

[0084] Table 2: PKD1 ultra-long fragment PCR amplification system

[0085]

[0086] Table 3: PKD1 ultra-long fragment PCR amplification program

[0087]

[0088] (2) After the long fragment amplification is completed, take 5 μL of the amplification product, add 1 μL of 6× Loading Buffer, mix well and perform 0.4% agarose gel electrophoresis to observe the amplification result. The result is as Figure 2 shown. The bp numbers of the four amplified long fragments of PKD1 are: Amplicon 1: 2813 bp, Amplicon 2: 8573 bp, Amplicon 3: 7092 bp, Amplicon 4: 8460 bp. According to the position of the Marker, it is confirmed that the four amplicons of PKD1 are specifically recognized and amplified by the amplification primer set of the present invention.

[0089] For the gel extraction of PCR products, use the Novoprotein FastPure Gel DNA Extraction Mini Kit (DC301-01) and perform the gel extraction operation according to the kit instructions.

[0090] (3) Use a Qubit4.0 instrument to measure the concentration of the recovered product. According to the formula, the genomic quality (ng) of the purified amplification product = (X * N * 660 * 10 -6 ) * 10 for sample mixing. The specific addition amounts are shown in Table 4. In Table 4, "Sampling amount (ng)*" represents the ng number of sampling calculated according to the amplicon fragment length and the fixed molar value. After mixing evenly, it is reserved for use.

[0091] Table 4: Mixed addition amounts of amplicons 1-4 and genomic DNA

[0092]

[0093]

[0094] 3. Library construction, liquid hybridization capture and high-throughput sequencing

[0095] The library construction was carried out using AngTruth-seq TM EZ DNA Library Preparation Module (Zhongke Gene) was used for preparation. The original gDNA sample and the sample after mixing with Example 2 were centrifuged and placed on an ice box. 100 ng was taken respectively for library construction. The library was quantitatively tested for quality using a fluorescence quantifier (Qubit TM 4.0 Fluorometer).

[0096] Figure 3 It is a quality inspection diagram of the library constructed using the Panel related to renal cyst disease. As can be seen from Figure 3 In the detection by the fully automatic nucleic acid and protein analysis system, the proportion of fragments with lengths of 150 - 800 bp in the library constructed in the examples was ≥ 60%, the proportion of fragments with lengths less than 150 bp was < 5%, there was no obvious large fragment trailing (there was a peak when > 1 Kb), and the concentration and total amount met the requirements for loading onto the T7 sequencer. This indicates that the quality of the library constructed in this example is qualified.

[0097] Using AngTruth-seq TM Target Module (Zhongke Gene) for liquid hybridization capture. After capture, the library was PCR-enriched and sequenced on an MGI T7 sequencer. The original raw data FASTQ was quality-controlled, adapter-trimmed, and low-quality data filtered using fastp (version 0.23.2).

[0098] Figure 4 It is a diagram of the coverage depth and coverage rate obtained by MGIT7 sequencing and bioinformatics analysis statistics of the library constructed for the Panel related to renal cyst disease. As can be seen from Figure 4 The average coverage depth of exons 1 - 34 reached 450×, and the coverage rate of all exons was 100%. This result indicates that all exon regions covered by the Panel were fully sequenced, and the high coverage depth and complete coverage rate ensured the high quality and reliability of the sequencing data, providing a solid foundation for subsequent variant detection. In particular, the high average coverage depth of exons 1 - 34 further highlighted the advantages of the amplification primer set and mutation detection method adopted in this study in accurately detecting variants in this region of the genome, helping to improve the accuracy and sensitivity of detection, and thus more effectively identifying potential pathogenic variants related to renal cyst disease.

[0099] 4. Sequencing data analysis

[0100] The filtered FASTQ was aligned to the human reference genome (build hg19) using bwa-mem (version 2.2.1) to generate the alignment file. The alignment file was processed and quality controlled using samblaster (version 0.1.26), samtools (version 1.16.1), and mosdepth (version 0.3.3). The processed alignment file was used for variant detection using gatk (version 4.3.0.0). The variant detection results were filtered and quality controlled using bcftools (version 1.16). The variant detection results were annotated using snpeff (version 5.0) and annovar (version 2020-06-08).

[0101] The sequencing results are as Figures 5 - 6 shown Figure 5 the IGV view of the gene detection results after amplifying the PKD1 gene using the amplification primer set of the present invention as primers in the examples. The positions marked by the boxes are the specific sites of the true gene; among them Figure 5 (a) shows the gene detection of the exon 1 region in the PKD1 gene; Figure 5 (b) shows the gene detection of the exon 2-14 regions in the PKD1 gene; Figure 5 (c) shows the gene detection of the exon 15-21 regions in the PKD1 gene; Figure 5 (d) shows the gene detection of the exon 22-34 regions in the PKD1 gene; Figure 6 is the Sanger sequencing verification result of the specific sites of the true gene in the gene obtained after amplifying the PKD1 gene using the amplification primer set of the present invention as primers in the examples. The positions marked by the boxes are the specific sites of the true gene; among them Figure 6 (a) shows the sequencing verification result of the exon 1 region in the PKD1 gene; Figure 6 (b) shows the sequencing verification result of the exon 2-14 regions in the PKD1 gene; Figure 6 (c) shows the sequencing verification result of the exon 15-21 regions in the PKD1 gene; Figure 6 (d) shows the sequencing verification result of the exon 22-34 regions in the PKD1 gene.

[0102] Figures 5 - 6 Compared with Figure 1 the sequence alignment of the true gene and the false gene of exons 1-34 of the PKD1 gene in, it is shown that in this example, by using the amplification primer set of the present invention to amplify the PKD1 gene, which is the gene for renal cyst disease in the collected human samples, the obtained results are all true gene amplification results.

[0103] As demonstrated by the above embodiments, the PKD1 ultra-long fragment PCR amplification primers and the method for detecting mutations in the PKD1 gene of renal cyst disease of the present invention can distinguish between true and false PKD1 genes, greatly improve the diagnostic accuracy rate, simplify the operation process, improve the timeliness, and increase the detection throughput.

[0104] For the same or similar parts among the various embodiments in this specification, reference can be made to each other. Each embodiment focuses on the differences from other embodiments.

[0105] The above is only the specific implementation manner of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed in the present application should be covered by the protection scope of the present application. Therefore, the protection scope of the present application shall be subject to the protection scope of the claims.

Claims

1. A primer set for amplifying the gene PKD1 of polycystic kidney disease, characterized in that, The amplification primer set includes 4 pairs of primers that specifically amplify exons 1 to 34 of the PKD1 gene: The primer for amplifying exon 1 of the PKD1 gene, its forward primer is as shown in SEQ ID NO:1, and its reverse primer is as shown in SEQ ID NO:2; The primer for amplifying exons 2-14 of the PKD1 gene, its forward primer is as shown in SEQ ID NO:3, and its reverse primer is as shown in SEQ ID NO:4; The primer for amplifying exons 15-21 of the PKD1 gene, its forward primer is as shown in SEQ ID NO:5, and its reverse primer is as shown in SEQ ID NO:6; The primer for amplifying exons 22-34 of the PKD1 gene, its forward primer is as shown in SEQ ID NO:7, and its reverse primer is as shown in SEQ ID NO:

8.

2. The amplification primer set for the gene PKD1 of renal cyst disease according to claim 1, characterized in that, The size of the PDK1 gene amplification fragment amplified by the primer for amplifying exon 1 of the PKD1 gene is 2813bp; The size of the PDK1 gene amplification fragment amplified by the primer for amplifying exons 2-14 of the PKD1 gene is 8573bp; The size of the PDK1 gene amplification fragment amplified by the primer for amplifying exons 15-21 of the PKD1 gene is 7092bp; The size of the PDK1 gene amplification fragment amplified by the primer for amplifying exons 22-34 of the PKD1 gene is 8460bp.

3. The amplification primer set for the gene PKD1 of polycystic kidney disease according to claim 1, characterized in that, The primers of SEQ ID NO:1-8 have a GC content of 40%-60% and do not have a repetitive sequence of more than 4 consecutive identical bases.

4. The amplification primer set for the kidney cyst disease gene PKD1 according to claim 1, wherein The average coverage depth of exons 1-34 obtained by amplification reaches 450×, and the coverage rate of all exons is 100%.

5. A method for detecting mutations in the gene PKD1 for polycystic kidney disease as described in claim 1, characterized in that, It includes the following steps: (1) Obtain genomic DNA; (2) Perform ultra-long fragment PCR amplification on the genomic DNA in step (1) with the amplification primer set, detect by electrophoresis and purify the target fragment; (3) Mix the purified amplification product obtained in step (2) with genomic DNA, construct a sequencing library and perform high-throughput sequencing through target region capture; (4) Analyze the sequencing data and screen for pathogenic PKD1 gene mutations.

6. The method for detecting the mutation of the gene PKD1 for renal cyst disease according to claim 5, characterized in that, In step (2), the ultra-long fragment amplification reaction conditions are as follows: pre-denature at 98°C for 3 min, then perform 35 cycles, each cycle is denaturation at 98°C for 15 s, annealing at 68°C for 20 s, extension at 72°C for 9 min, and finally perform a thorough extension at 72°C for 9 min, and store at 4°C.

7. The method for detecting mutations of the gene PKD1 for polycystic kidney disease according to claim 5, characterized in that, In step (3), the addition amount of the purified amplification product is calculated according to the following formula: the genomic quality (ng) of the purified amplification product = (X * N * 660 * 10 -6 ) * 10, where X is the femtomole number of the recovered fragment; N is the length of the recovered fragment.

8. The method for detecting mutations of the gene PKD1 for renal cyst disease according to claim 5, characterized in that, In step (3), use a Panel related to renal cyst disease to perform liquid hybridization capture for target region capture of the library.

9. The method for detecting mutations of the gene PKD1 for polycystic kidney disease according to claim 5, characterized in that, In step (4), identify pathogenic PKD1 gene mutations through sequence alignment, variant detection and functional annotation.

10. Use of an amplification primer set for the renal cyst disease gene PKD1 as described in claim 1 in the detection of mutations in the renal cyst disease gene PKD1.

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